This study provides Class I evidence that 4 and 8 mg/day doses of adjunctive perampanel are effective and tolerated in reducing partial-onset seizures.

Microglia can be isolated with high purity but low yield by shaking off loosely adherent cells from mixed glial cultures. Here we describe a new technique for isolating microglia with an average yield close to 2,000,000 microglial cells/mouse pup, more than five times higher than that of the shaking method. Confluent mixed glial cultures are subjected to mild trypsinization (0.05-0.12%) in the presence of 0.2-0.5 mM EDTA and 0.5-0.8 mM Ca2+. This results in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving undisturbed a population of firmly attached cells identified as >98% microglia. These almost pure microglial preparations can be kept in culture for weeks and show proliferation and phagocytosis. Treatment with macrophage colony-stimulating factor and lipopolysaccharide, alone or in the presence of interferon gamma, induces typical microglial responses in terms of proliferation, morphological changes, nuclear factor-kappaB translocation, NO, and tumor necrosis alpha release and phagocytosis. This method allows for the preparation of highly enriched mouse or rat microglial cultures with ease and reproducibility. Because of its high yield, it can be especially convenient when high amounts of microglial protein/mRNA are required or in cases in which the starting material is limited, such as microglial cultures from transgenic animals.

Idiopathic focal epilepsy (IFE) with rolandic spikes is the most common childhood epilepsy, comprising a phenotypic spectrum from rolandic epilepsy (also benign epilepsy with centrotemporal spikes, BECTS) to atypical benign partial epilepsy (ABPE), Landau-Kleffner syndrome (LKS) and epileptic encephalopathy with continuous spike and waves during slow-wave sleep (CSWS). The genetic basis is largely unknown. We detected new heterozygous mutations in GRIN2A in 27 of 359 affected individuals from 2 independent cohorts with IFE (7.5%; P = 4.83 × 10(-18), Fisher's exact test). Mutations occurred significantly more frequently in the more severe phenotypes, with mutation detection rates ranging from 12/245 (4.9%) in individuals with BECTS to 9/51 (17.6%) in individuals with CSWS (P = 0.009, Cochran-Armitage test for trend). In addition, exon-disrupting microdeletions were found in 3 of 286 individuals (1.0%; P = 0.004, Fisher's exact test). These results establish alterations of the gene encoding the NMDA receptor NR2A subunit as a major genetic risk factor for IFE.

The majority of epilepsies are focal in origin, with seizures emanating from one brain region. Although focal epilepsies often arise from structural brain lesions, many affected individuals have normal brain imaging. The etiology is unknown in the majority of individuals, although genetic factors are increasingly recognized. Autosomal dominant familial focal epilepsy with variable foci (FFEVF) is notable because family members have seizures originating from different cortical regions. Using exome sequencing, we detected DEPDC5 mutations in two affected families. We subsequently identified mutations in five of six additional published large families with FFEVF. Study of families with focal epilepsy that were too small for conventional clinical diagnosis with FFEVF identified DEPDC5 mutations in approximately 12% of families (10/82). This high frequency establishes DEPDC5 mutations as a common cause of familial focal epilepsies. Shared homology with G protein signaling molecules and localization in human neurons suggest a role of DEPDC5 in neuronal signal transduction.

Epileptic encephalopathies are a phenotypically and genetically heterogeneous group of severe epilepsies accompanied by intellectual disability and other neurodevelopmental features1-6. Using next generation sequencing, we identified four different de novo mutations in KCNA2, encoding the potassium channel KV1.2, in six patients with epileptic encephalopathy (one mutation recurred three times independently). Four individuals presented with febrile and multiple afebrile, often focal seizure types, multifocal epileptiform discharges strongly activated by sleep, mild-moderate intellectual disability, delayed speech development and sometimes ataxia. Functional studies of the two mutations associated with this phenotype revealed an almost complete loss-of-function with a dominant-negative effect. Two further individuals presented with a different and more severe epileptic encephalopathy phenotype. They carried mutations inducing a drastic gain-of-function effect leading to permanently open channels. These results establish KCNA2 as a novel gene involved in human neurodevelopmental disorders by two different mechanisms, predicting either hyperexcitability or electrical silencing of KV1.2-expressing neurons.

Lafora progressive myoclonus epilepsy (LD) is a fatal autosomal recessive neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies. LD is caused by mutations in two genes, EPM2A and EPM2B, encoding respectively laforin, a dual-specificity protein phosphatase, and malin, an E3 ubiquitin ligase. Previously, we and others have suggested that the interactions between laforin and PTG (a regulatory subunit of type 1 protein phosphatase) and between laforin and malin are critical in the pathogenesis of LD. Here, we show that the laforin-malin complex downregulates PTG-induced glycogen synthesis in FTO2B hepatoma cells through a mechanism involving ubiquitination and degradation of PTG. Furthermore, we demonstrate that the interaction between laforin and malin is a regulated process that is modulated by the AMP-activated protein kinase (AMPK). These findings provide further insights into the critical role of the laforin-malin complex in the control of glycogen metabolism and unravel a novel link between the energy sensor AMPK and glycogen metabolism. These data advance our understanding of the functional role of laforin and malin, which hopefully will facilitate the development of appropriate LD therapies.

Dravet syndrome is a severe epilepsy syndrome characterized by infantile onset of therapy-resistant, fever-sensitive seizures followed by cognitive decline. Mutations in SCN1A explain about 75% of cases with Dravet syndrome; 90% of these mutations arise de novo. We studied a cohort of nine Dravet-syndrome-affected individuals without an SCN1A mutation (these included some atypical cases with onset at up to 2 years of age) by using whole-exome sequencing in proband-parent trios. In two individuals, we identified a de novo loss-of-function mutation in CHD2 (encoding chromodomain helicase DNA binding protein 2). A third CHD2 mutation was identified in an epileptic proband of a second (stage 2) cohort. All three individuals with a CHD2 mutation had intellectual disability and fever-sensitive generalized seizures, as well as prominent myoclonic seizures starting in the second year of life or later. To explore the functional relevance of CHD2 haploinsufficiency in an in vivo model system, we knocked down chd2 in zebrafish by using targeted morpholino antisense oligomers. chd2-knockdown larvae exhibited altered locomotor activity, and the epileptic nature of this seizure-like behavior was confirmed by field-potential recordings that revealed epileptiform discharges similar to seizures in affected persons. Both altered locomotor activity and epileptiform discharges were absent in appropriate control larvae. Our study provides evidence that de novo loss-of-function mutations in CHD2 are a cause of epileptic encephalopathy with generalized seizures.

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