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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. ANN NEUROL 2012;72:807–815

The genetic architecture of common epilepsies is largely unknown. HCNs are excellent epilepsy candidate genes because of their fundamental neurophysiological roles. Screening in subjects with febrile seizures and genetic epilepsy with febrile seizures plus revealed that 2.4% carried a common triple proline deletion (delPPP) in HCN2 that was seen in only 0.2% of blood bank controls. Currents generated by mutant HCN2 channels were ~35% larger than those of controls; an effect revealed using automated electrophysiology and an appropriately powered sample size. This is the first association of HCN2 and familial epilepsy, demonstrating gain of function of HCN2 current as a potential contributor to polygenic epilepsy.

We have systematically screened EMS-mutagenized Drosophila for embryonic lethal strains with defects in glutamatergic synaptic transmission. Surprisingly, this screen led to the identification of several alleles with missense mutations in highly conserved regions of Dgad1. Analysis of these gad mutants reveals that they are paralyzed owing to defects in glutamatergic transmission at the neuromuscular junction. Further electrophysiological and immunohistochemical examination reveals that these mutants have greatly reduced numbers of postsynaptic glutamate receptors in an otherwise morphologically normal synapse. By overexpressing wild-type Dgad1 in selected neurons, we show that GAD is specifically required in the presynaptic neuron to induce a postsynaptic glutamate receptor field, and that the level of postsynaptic receptors is closely dependent on presynaptic GAD function. These data demonstrate that GAD plays an unexpected role in glutamatergic synaptogenesis.

The stoned locus of Drosophila melanogaster encodes two novel proteins, stonedA (STNA) and stonedB (STNB), both of which are expressed in the nervous system. Flies with defects at the stoned locus have abnormal behavior and altered synaptic transmission. Genetic interactions, in particular with the shibire (dynamin) mutation, indicated a presynaptic function for stoned and suggested an involvement in vesicle cycling. Immunological studies revealed colocalization of the stoned proteins at the neuromuscular junction with the integral synaptic vesicle protein synaptotagmin (SYT). We show here that stoned interacts genetically with synaptotagmin to produce a lethal phenotype. The STNB protein is found by co-immunoprecipitation to be associated with synaptic vesicles, and glutathione S-transferase pull-downs demonstrate an in vitro interaction between the micro2-homology domain of STNB and the C2B domain of the SYTI isoform. The STNA protein is also found in association with vesicles, and it too exhibits an in vitro association with SYTI. However, we find that the bulk of STNA is in a nonmembranous fraction. By using the shibire mutant to block endocytosis, STNB is shown to be present on some synaptic vesicles before exocytosis. However, STNB is not associated with all synaptic vesicles. We hypothesize that STNB specifies a subset of synaptic vesicles with a role in the synaptic vesicle cycle that is yet to be determined.

Objective: To understand the molecular basis and differential penetrance of febrile seizures and absence seizures in patients with the g2(R43Q) GABA A receptor mutation.Methods: Spike-and-wave discharges and thermal seizure susceptibility were measured in heterozygous GABA A g2 knock-out and GABA A g2(R43Q) knock-in mice models crossed to different mouse strains.Results: By comparing the GABA A g2 knock-out with the GABA A g2(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. Neurology Family members harboring the GABA A g2(R43Q) mutation display multiple seizure types, incomplete penetrance, and variable seizure severity, 1,2 a common feature of genetic epilepsies. 3To predict clinical outcomes for patients based on their personal genomes, it is important that we develop a conceptual framework explaining the genetic and molecular basis of heterogeneity. Animal models of epilepsy, based on human mutations, provide a means of investigating this. The knock-in mouse model based on the GABA A g2(R43Q) mutation recapitulates the 2 major phenotypes seen in family members, including febrile seizures and typical absence seizures. 4,5 However, the molecular mechanisms causing epilepsy in this model are unclear. In vitro studies suggest that the deficit could be through haploinsufficiency (loss-of-function) or a dominant impact of the mutated protein. 6 To investigate this, we compared the seizure phenotypes of heterozygous Gabrg2 knock-out mice with those of knock-in mice.Penetrance is relatively low for absence seizures in the GABA A g2(R43Q) family. 1,2 By backcrossing the GABA A g2(R43Q) knock-in mouse to strains with different seizure susceptibility, we have demonstrated that the spike-wave phenotype requires additional susceptibility alleles for full expression, 5,7 potentially explaining the low penetrance of absence in the family. In contrast, febrile seizures segregate as a more highly penetrant autosomal dominant trait, which suggests that background genetics have less impact.1,2 Here we investigate the influence of genetic background on thermal seizure and spike-wave susceptibility in the knock-in mouse model. METHODS Mice. All experiments were approved by the Animal Ethics Committee at the Florey Institute of Neuroscience and Mental Health (09-046). Genotyping of the GABA...

The cuticular melanization phenotype of black flies is rescued by h-alanine, but h-alanine production, by aspartate decarboxylation, was reported to be normal in assays of black mutants, and although black/Dgad2 is expressed in the lamina, the first optic ganglion, no electroretinogram (ERG) or other visual defect has been demonstrated in black flies. The purpose of this study was to investigate the black gene, and protein, in black 1 mutants of Drosophila melanogaster in order to resolve the apparent paradox of the black phenotype. Using black 1 mutant flies we show that (1) aspartate decarboxylase activity is significantly reduced in adults and at puparium formation, consistent with defects in cuticular and non-cuticular processes, (2) that the black 1 mutation is a frameshift, and black 1 flies are nulls for the black/ DGAD2 protein, and (3) that behavioural experiments using Buridan's paradigm, demonstrate that black responds abnormally to visual cues. No ERG, or target recognition defects can be demonstrated suggesting a problem with higher order visual functions in black mutants. D

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 Ih 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. AbbreviationsFS, febrile seizures; GAERS, generalized Absence Epilepsy Rat from Strasburg; GGE, genetic generalized epilepsy; HCN, hyperpolarization-activated cyclic nucleotide-gated channels; HCN2 KO, HCN2 knockout; IGE, idiopathic generalized epilepsy; Ih, current mediated by HCN; SWD, spike-and-wave discharge; TRIP8b, TPR-containing Rab8b interacting protein; WAG/Rij, Wistar Albino Glaxo rats, bred in Rijswijk IntroductionEpilepsy, with a lifetime prevalence of 3%, is a common and serious neurological disorder with significant associated morbidity and mortality. Approximately a third of sufferers do not respond satisfactorily to current treatments. Understanding the molecular basis of epilepsy is key to the development of new approaches to therapy that have improved efficacy and tolerability. Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are one candidate group of ion channels that has received particular attention in recent years. There are four known HCN channels encoded by HCN1, HCN2, HCN3 & HCN4 (Ludwig et al., 1998;Santoro and Tibbs, 1999), with each having distinct kinetic and voltage characteristics as well as cAMP sensitivity (reviewed in Biel et al., 2009). HCN channels are expressed in both neurons and myocytes (Biel et al., 2009). In the heart they conduct If which is critical for the pacemaker function underlying cardiac rhythm (reviewed in Mangoni and Nargeot, 2008). A pacemaker role is thought to be important for the analogous Ih current in the brain. Epilepsy is a group of neurological disorders characterized by the occurrence of recurrent spontaneous seizures, the basis of which is hypersynchronous network activity, typically driven by a pacemaker process. It is, therefore, not surprising that evidence for ...