WONOEP appraisal: New genetic approaches to study epilepsy

New genetic investigation techniques, including next-generation sequencing, epigenetic profiling, cell lineage mapping, targeted genetic manipulation of specific neuronal cell types, stem cell reprogramming, and optogenetic manipulations within epileptic networks are progressively unraveling the mysteries of epileptogenesis and ictogenesis. These techniques have opened new avenues to discover the molecular basis of epileptogenesis and to study the physiologic effects of mutations in epilepsy associated genes on a multilayer level, from cells to circuits. This manuscript reviews recently published applications of these new genetic technologies in the study of epilepsy, as well as work presented by the authors at the genetic session of the XII Workshop on the Neurobiology of Epilepsy (WONOEP 2013) in Quebec, Canada. Next-generation sequencing is providing investigators with an unbiased means to assess the molecular causes of sporadic forms of epilepsy and has revealed the complexity and genetic heterogeneity of sporadic epilepsy disorders. To assess the functional impact of mutations in these newly identified genes on specific neuronal cell types during brain development, new modeling strategies in animals, including conditional genetics in mice and in utero knock-down approaches, are enabling functional validation with exquisite cell-type and temporal specificity. In addition, optogenetics, using cell-type–specific Cre recombinase driver lines, is enabling investigators to dissect networks involved in epilepsy. In addition, genetically encoded cell-type labeling is providing new means to assess the role of the nonneuronal components of epileptic networks such as glial cells. Furthermore, beyond its role in revealing coding variants involved in epileptogenesis, next-generation sequencing can be used to assess the epigenetic modifications that lead to sustained network hyperexcitability in epilepsy, including methylation changes in gene promoters and noncoding ribonucleic acid (RNA) involved in modifying gene expression following seizures. In addition, genetically based bioluminescent reporters are providing new opportunities to assess neuronal activity and neurotransmitter levels both in vitro and in vivo in the context of epilepsy. Finally, genetically rederived neurons generated from patient induced pluripotent stem cells and genetically modified zebrafish have become high-throughput means to investigate disease mechanisms and potential new therapies. Genetics has changed the field of epilepsy research considerably, and is paving the way for better diagnosis and therapies for patients with epilepsy

WONOEP appraisal: New genetic approaches to study epilepsy

New genetic investigation techniques, including next-generation sequencing, epigenetic profiling, cell lineage mapping, targeted genetic manipulation of specific neuronal cell types, stem cell reprogramming, and optogenetic manipulations within epileptic networks are progressively unraveling the mysteries of epileptogenesis and ictogenesis. These techniques have opened new avenues to discover the molecular basis of epileptogenesis and to study the physiologic effects of mutations in epilepsy associated genes on a multilayer level, from cells to circuits. This manuscript reviews recently published applications of these new genetic technologies in the study of epilepsy, as well as work presented by the authors at the genetic session of the XII Workshop on the Neurobiology of Epilepsy (WONOEP 2013) in Quebec, Canada. Next-generation sequencing is providing investigators with an unbiased means to assess the molecular causes of sporadic forms of epilepsy and has revealed the complexity and genetic heterogeneity of sporadic epilepsy disorders. To assess the functional impact of mutations in these newly identified genes on specific neuronal cell types during brain development, new modeling strategies in animals, including conditional genetics in mice and in utero knock-down approaches, are enabling functional validation with exquisite cell-type and temporal specificity. In addition, optogenetics, using cell-type–specific Cre recombinase driver lines, is enabling investigators to dissect networks involved in epilepsy. In addition, genetically encoded cell-type labeling is providing new means to assess the role of the nonneuronal components of epileptic networks such as glial cells. Furthermore, beyond its role in revealing coding variants involved in epileptogenesis, next-generation sequencing can be used to assess the epigenetic modifications that lead to sustained network hyperexcitability in epilepsy, including methylation changes in gene promoters and noncoding ribonucleic acid (RNA) involved in modifying gene expression following seizures. In addition, genetically based bioluminescent reporters are providing new opportunities to assess neuronal activity and neurotransmitter levels both in vitro and in vivo in the context of epilepsy. Finally, genetically rederived neurons generated from patient induced pluripotent stem cells and genetically modified zebrafish have become high-throughput means to investigate disease mechanisms and potential new therapies. Genetics has changed the field of epilepsy research considerably, and is paving the way for better diagnosis and therapies for patients with epilepsy

Case report: Off-label use of low-dose perampanel in a 25-month-old girl with a pathogenic SYNGAP1 variant

IntroductionPreclinical studies in a mouse model have shown that SYNGAP1 haploinsufficiency results in an epilepsy phenotype with excessive GluA2-AMPA insertion specifically on the soma of fast-spiking parvalbumin-positive interneurons associated with significant dysfunction of cortical gamma homeostasis that was rescued by perampanel (PER), an AMPA receptor blocker. In this single case, we aimed to investigate the presence of dysregulated cortical gamma in a toddler with a pathogenic SYNGAP1 variant and report on the effect of low-dose PER on electroencephalogram (EEG) and clinical profile.MethodsClinical data from physician's clinic notes; genetic testing reports; developmental scores from occupational therapy, physical therapy, speech and language therapy evaluations; and applied behavioral analysis reports were reviewed. Developmental assessments and EEG analysis were done pre- and post-PER.ResultsClinically, the patient showed improvements in the developmental profile and sleep quality post-PER. EEG spectral power analysis in our patient revealed a loss of gamma power modulation with behavioral-state transitions similar to what was observed in Syngap1+/− mice. Furthermore, the administration of low-dose PER rescued the dysfunctional cortical gamma homeostasis, similar to the preclinical study. However, as in the epileptic mice, PER did not curb epileptiform discharges or clinical seizures.ConclusionSimilar to the Syngap1+/− mice, cortical gamma homeostasis was dysregulated in the patient. This dysfunction was rescued by PER. These encouraging results necessitate further validation of gamma dysregulation as a potential translational EEG biomarker in SYNAP1-DEE. Low-dose PER can be explored as a therapeutic option through clinical trials

Neonatal seizures: impact on neurodevelopmental outcomes.

Neonatal period is the most vulnerable time for the occurrence of seizures, and neonatal seizures often pose a clinical challenge both for their acute management and frequency of associated long-term co-morbidities. Etiologies of neonatal seizures are known to play a primary role in the AED responsiveness and the long-term sequelae. Recent studies have suggested that burden of acute recurrent seizures in neonates may also impact chronic outcomes independent of the etiology. However, not many studies, either clinical or pre-clinical, have addressed the long-term outcomes of neonatal seizures in an etiology-specific manner. In this review, we briefly review the available clinical and pre-clinical research for long-term outcomes following neonatal seizures. As the most frequent cause of acquired neonatal seizures, we focus on the studies evaluating long-term effects of HIE seizures with the goal to evaluate: 1) what parameters evaluated during acute stages of neonatal seizures can reliably be used to predict long-term outcomes?, and 2) what clinical and pre-clinical data are available to help determine the importance of etiology vs. seizure burdens for long-term sequelae

Sex-Dependent Signaling Pathways Underlying Seizure Susceptibility and the Role of Chloride Cotransporters

Seizure incidence, severity, and antiseizure medication (ASM) efficacy varies between males and females. Differences in sex-dependent signaling pathways that determine network excitability may be responsible. The identification and validation of sex-dependent molecular mechanisms that influence seizure susceptibility is an emerging focus of neuroscience research. The electroneutral cation-chloride cotransporters (CCCs) of the SLC12A gene family utilize Na+-K+-ATPase generated electrochemical gradients to transport chloride into or out of neurons. CCCs regulate neuronal chloride gradients, cell volume, and have a strong influence over the electrical response to the inhibitory neurotransmitter GABA. Acquired or genetic causes of CCCs dysfunction have been linked to seizures during early postnatal development, epileptogenesis, and refractoriness to ASMs. A growing number of studies suggest that the developmental expression of CCCs, such as KCC2, is sex-dependent. This review will summarize the reports of sexual dimorphism in epileptology while focusing on the role of chloride cotransporters and their associated modulators that can influence seizure susceptibility

Neonatal stroke in mice causes long-term changes in neuronal notch-2 expression that may contribute to prolonged injury

Background and Purpose: Notch receptors (1–4) are membrane proteins that, on ligand stilumation, release their cytoplasmic domains to serve as transcription factors. Notch-2 promotes proliferation both during development and cancer, but its role in response to ischemic injury is less well understood. The purpose of this study was to understand whether Notch-2 is induced after neonatal stroke and to investigate its functional relevance.Methods: P12 CD1 mice were subjected to permanent unilateral (right-sided) double ligation of the common carotid artery.Results: Neonatal ischemia induces a progressive brain injury with prolonged apoptosis and Notch-2 up-regulation. Notch-2 expression was induced shortly after injury in hippocampal areas with elevated c-fos activation and increased cell death. Long-term induction of Notch-2 also occurred in CA1 and CA3 in and around areas of cell death, and had a distinct pattern of expression as compared to Notch-1. In vitro oxygen glucose deprivation treatment showed a similar increase in Notch-2 in apoptotic cells. In vitro gain of function experiments, using an active form of Notch-2, show that Notch-2 induction is neurotoxic to a comparable extent as oxygen glucose deprivation treatment.Conclusions: These results suggest that Notch-2 up-regulation after neonatal ischemia is detrimental to neuronal survival

Temporal- and Location-Specific Alterations of the GABA Recycling System in Mecp2 KO Mouse Brains

Rett syndrome (RTT), associated with mutations in methyl-CpG-binding protein 2 (Mecp2), is linked to diverse neurological symptoms such as seizures, motor disabilities, and cognitive impairments. An altered GABAergic system has been proposed as one of many underlying pathologies of progressive neurodegeneration in several RTT studies. This study for the first time investigated the temporal- and location-specific alterations in the expression of γ-amino butyric acid (GABA) transporter 1 (GAT-1), vesicular GABA transporter (vGAT), and glutamic acid decarboxylase 67kD (GAD67) in wild type (WT) and knockout (KO) mice in the Mecp2 m1.1Bird/y mouse model of RTT. Immunohistochemistry (IHC) co-labeling of GAT-1 with vGAT identified GABAergic synapses that were quantitated for mid-sagittal sections in the frontal cortex (FC), hippocampal dentate gyrus (DG), and striatum (Str). An age-dependent increase in the expression of synaptic GABA transporters, GAT-1, and vGAT, was observed in the FC and DG in WT brains. Mecp2 KO mice showed a significant alteration in this temporal profile that was location-specific, only in the FC. GAD67-positive cell densities also showed an age-dependent increase in the FC, but a decrease in the DG in WT mice. However, these densities were not significantly altered in the KO mice in the regions examined in this study. Therefore, the significant location-specific downregulation of synaptic GABA transporters in Mecp2 KO brains with unaltered densities of GAD67-positive interneurons may highlight the location-specific synaptic pathophysiology in this model of RTT

Rett Syndrome and CDKL5 Deficiency Disorder: From Bench to Clinic

Rett syndrome (RTT) and CDKL5 deficiency disorder (CDD) are two rare X-linked developmental brain disorders with overlapping but distinct phenotypic features. This review examines the impact of loss of methyl-CpG-binding protein 2 (MeCP2) and cyclin-dependent kinase-like 5 (CDKL5) on clinical phenotype, deficits in synaptic- and circuit-homeostatic mechanisms, seizures, and sleep. In particular, we compare the overlapping and contrasting features between RTT and CDD in clinic and in preclinical studies. Finally, we discuss lessons learned from recent clinical trials while reviewing the findings from pre-clinical studies

A companion to the preclinical common data elements and case report forms for neuropathology studies in epilepsy research. A report of the TASK3 WG2 Neuropathology Working Group of the ILAE/AES Joint Translational Task Force

The International League Against Epilepsy/American Epilepsy Society (ILAE/AES) Joint Translational Task Force initiated the TASK3 working group to create common data elements (CDEs) for various aspects of preclinical epilepsy research studies, which could help improve the standardization of experimental designs. This article addresses neuropathological changes associated with seizures and epilepsy in rodent models of epilepsy. We discuss CDEs for histopathological parameters for neurodegeneration, changes in astrocyte morphology and function, mechanisms of inflammation, and changes in the blood-brain barrier and myelin/oligodendrocytes resulting from recurrent seizures in rats and mice. We provide detailed CDE tables and case report forms (CRFs), and with this companion manuscript, we discuss the rationale and methodological aspects of individual neuropathological examinations. The CDEs, CRFs, and companion paper are available to all researchers, and their use will benefit the harmonization and comparability of translational preclinical epilepsy research. The ultimate hope is to facilitate the development of rational therapy concepts for treating epilepsies, seizures, and comorbidities and the development of biomarkers assessing the pathological state of the disease