Jennifer C. Wong

SCN3A deficiency associated with increased seizure susceptibility

Mutations in voltage-gated sodium channels expressed highly in the brain (SCN1A, SCN2A, SCN3A, and SCN8A) are responsible for an increasing number of epilepsy syndromes. In particular, mutations in the SCN3A gene, encoding the pore-forming Nav1.3 α subunit, have been identified in patients with focal epilepsy. Biophysical characterization of epilepsy-associated SCN3A variants suggests that both gain- and loss-of-function SCN3A mutations may lead to increased seizure susceptibility. In this report, we identified a novel SCN3A variant (L247P) by whole exome sequencing of a child with focal epilepsy, developmental delay, and autonomic nervous system dysfunction. Voltage clamp analysis showed no detectable sodium current in a heterologous expression system expressing the SCN3A-L247P variant. Furthermore, cell surface biotinylation demonstrated a reduction in the amount of SCN3A-L247P at the cell surface, suggesting the SCN3A-L247P variant is a trafficking-deficient mutant. To further explore the possible clinical consequences of reduced SCN3A activity, we investigated the effect of a hypomorphic Scn3a allele (Scn3aHyp) on seizure susceptibility and behavior using a gene trap mouse line. Heterozygous Scn3a mutant mice (Scn3a+/Hyp) did not exhibit spontaneous seizures nor were they susceptible to hyperthermia-induced seizures. However, they displayed increased susceptibility to electroconvulsive (6Hz) and chemiconvulsive (flurothyl and kainic acid) induced seizures. Scn3a+/Hyp mice also exhibited deficits in locomotor activity and motor learning. Taken together, these results provide evidence that loss-of-function of SCN3A caused by reduced protein expression or deficient trafficking to the plasma membrane may contribute to increased seizure susceptibility.

The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo.

Huperzine A provides robust and sustained protection against induced seizures in Scn1a mutant mice

De novo loss-of-function mutations in the voltage-gated sodium channel (VGSC) SCN1A (encoding Nav1.1) are the main cause of Dravet syndrome (DS), a catastrophic early-life encephalopathy associated with prolonged and recurrent early-life febrile seizures (FSs), refractory afebrile epilepsy, cognitive and behavioral deficits, and a 15–20% mortality rate. SCN1A mutations also lead to genetic epilepsy with febrile seizures plus (GEFS+), which is an inherited disorder characterized by early-life FSs and the development of a range of adult epilepsy subtypes. Current antiepileptic drugs often fail to protect against the severe seizures and behavioral and cognitive deficits found in patients with SCN1A mutations. To address the need for more efficacious treatments for SCN1A-derived epilepsies, we evaluated the therapeutic potential of Huperzine A, a naturally occurring reversible acetylcholinesterase inhibitor. In CF1 mice, Hup A (0.56 or 1 mg/kg) was found to confer protection against 6 Hz-, pentylenetetrazole (PTZ)-, and maximal electroshock (MES)-induced seizures. Robust protection against 6 Hz-, MES-, and hyperthermia-induced seizures was also achieved following Hup A administration in mouse models of DS (Scn1a+/−) and GEFS+ (Scn1aRH/+). Furthermore, Hup A-mediated seizure protection was sustained during 3 weeks of daily injections in Scn1aRH/+ mutants. Finally, we determined that muscarinic and GABAA receptors play a role in Hup A-mediated seizure protection. These findings indicate that Hup A might provide a novel therapeutic strategy for increasing seizure resistance in DS and GEFS+, and more broadly, in other forms of refractory epilepsy.

GPR37L1 modulates seizure susceptibility: Evidence from mouse studies and analyses of a human GPR37L1 variant

Progressive myoclonus epilepsies (PMEs) are disorders characterized by myoclonic and generalized seizures with progressive neurological deterioration. While several genetic causes for PMEs have been identified, the underlying causes remain unknown for a substantial portion of cases. Here we describe several affected individuals from a large, consanguineous family presenting with a novel PME in which symptoms begin in adolescence and result in death by early adulthood. Whole exome analyses revealed that affected individuals have a homozygous variant in GPR37L1 (c.1047G>T [Lys349Asn]), an orphan G protein-coupled receptor (GPCR) expressed predominantly in the brain. In vitro studies demonstrated that the K349N substitution in Gpr37L1 did not grossly alter receptor expression, surface trafficking or constitutive signaling in transfected cells. However, in vivo studies revealed that a complete loss of Gpr37L1 function in mice results in increased seizure susceptibility. Mice lacking the related receptor Gpr37 also exhibited an increase in seizure susceptibility, while genetic deletion of both receptors resulted in an even more dramatic increase in vulnerability to seizures. These findings provide evidence linking GPR37L1 and GPR37 to seizure etiology and demonstrate an association between a GPR37L1 variant and a novel progressive myoclonus epilepsy.

Selective targeting of Scn8a prevents seizure development in a mouse model of mesial temporal lobe epilepsy

We previously found that genetic mutants with reduced expression or activity of Scn8a are resistant to induced seizures and that co-segregation of a mutant Scn8a allele can increase survival and seizure resistance of Scn1a mutant mice. In contrast, Scn8a expression is increased in the hippocampus following status epilepticus and amygdala kindling. These findings point to Scn8a as a promising therapeutic target for epilepsy and raise the possibility that aberrant overexpression of Scn8a in limbic structures may contribute to some epilepsies, including temporal lobe epilepsy. Using a small-hairpin-interfering RNA directed against the Scn8a gene, we selectively reduced Scn8a expression in the hippocampus of the intrahippocampal kainic acid (KA) mouse model of mesial temporal lobe epilepsy. We found that Scn8a knockdown prevented the development of spontaneous seizures in 9/10 mice, ameliorated KA-induced hyperactivity, and reduced reactive gliosis. These results support the potential of selectively targeting Scn8a for the treatment of refractory epilepsy.

Illuminating the Cerebellum as a Potential Target for Treating Epilepsy.

Commentary Temporal lobe epilepsy (TLE) is most common form of refractory epilepsy, and mesial temporal lobe epilepsy (MTLE) is the most common subtype of TLE. MTLE is characterized by spontaneous seizures, behavioral abnormalities such as learning and memory deficits, and morphological changes in the hippocampus (e.g., neuron loss, mossy fiber sprouting) (1–3). At present, surgical resection of the seizure focus is the best treatment option; however, this invasive procedure can only be used in a subset of cases, identifying a critical need for the development of alternate treatments. Given the critical role of the hippocampus in TLE, this structure is considered the most obvious target for intervention. However, numerous projections extend to and from the hippocampus, suggesting that other brain regions might also make effective targets. In the current study, the cerebellum was evaluated as a potential therapeutic target for TLE. Several pieces of evidence provide support for the selection of the cerebellum. For example: 1) the cerebellum has been shown to influence hippocampal processing (4), and 2) direct connections between the cerebellum and hippocampus, via the midline of the cerebellum or nucleus fastigii, have been suggested as potential pathways for seizure regulation (5, 6). Optogenetics involves the use of light to excite or inhibit cells expressing channelrhodopsin or halorhodopsin, respectively. According to a recent review of optogenetics and epilepsy (7), a PubMed search of “optogenetics” conducted in August 2014 returned over 800 citations. As of June 2015, there are 1,201 citations for “optogenetics,” with 51 of these specifically for “optogenetics and epilepsy.” A recent study by Krook-Magnuson et al., which is the focus of this commentary, used optogenetics to evaluate the cerebellum as a potential therapeutic target in the well-established intrahippocampal kainic acid (KA) mouse model of MTLE. This model, generated by injecting a low dose of KA into the dorsal hippocampus, recapitulates many features of human MTLE, including spontaneous seizures that typically begin 3 to 4 weeks after KA administration (8). Krook-Magnuson and colleagues used a closed-loop seizure detection system (9) to trigger the delivery of light to different sites within the cerebellum following the development of spontaneous seizures in the MTLE mouse model. Light was administered in response to 50% of detected electrographic seizures in a randomized manner, thereby enabling each animal to serve as its own control. Using this approach, the authors first demonstrated that seizure duration could be altered by either activation or inhibition of parvalbuminexpressing (PV) neurons in the lateral cerebellar cortex. Specifically, stimulating PV neurons expressing the excitatory channelrhodopsin (results in activation of PV neurons) or the inhibitory halorhodopsin (results in inhibition of PV neurons) resulted in a significant reduction in seizure duration. While Cerebellar Directed Optogenetic Intervention Inhibits Spontaneous Hippocampal Seizures in a Mouse Model of Temporal Lobe Epilepsy.

Toward Routine Genetics-Based Diagnoses for the Epileptic Encephalopathies

Carvill GL, Heavin SB, Yendle SC, McMahon JM, O’Roak BJ, Cook J, Khan A, Dorschner MO, Weaver M, Calvert S, Malone S, Wallace G, Stanley T, Bye AME, Bleasel A, Howell KB, Kivity S, Mackay MT, Rodriguez-Casero V, Webster R, Korczyn A, Afawi Z, Zelnick N, Lerman-Sagie T, Lev D, Moller RS, Gill D, Andrade DM, Freeman JL, Sadleir LG, Shendure J, Berkovic SF, Scheffer IE, Mefford HC. Nat Genet 2013;45:825–831.

Noradrenergic Transmission at Alpha1-Adrenergic Receptors in the Ventral Periaqueductal Gray Modulates Arousal

BACKGROUND Dysregulation of arousal is symptomatic of numerous psychiatric disorders. Previous research has shown that the activity of dopamine (DA) neurons in the ventral periaqueductal gray (vPAG) tracks with arousal state, and lesions of vPAGDA cells increase sleep. However, the circuitry controlling these wake-promoting DA neurons is unknown. METHODS This study combined designer receptors exclusively activated by designer drugs (DREADDs), behavioral pharmacology, electrophysiology, and immunoelectron microscopy in male and female mice to elucidate mechanisms in the vPAG that promote arousal. RESULTS Activation of locus coeruleus projections to the vPAG or vPAGDA neurons induced by DREADDs promoted arousal. Similarly, agonist stimulation of vPAG alpha1-adrenergic receptors (α1ARs) increased latency to fall asleep, whereas α1AR blockade had the opposite effect. α1AR stimulation drove vPAGDA activity in a glutamate-dependent, action potential-independent manner. Compared with other dopaminergic brain regions, α1ARs were enriched on astrocytes in the vPAG, and mimicking α1AR transmission specifically in vPAG astrocytes via Gq-DREADDS was sufficient to increase arousal. In general, the wake-promoting effects observed were not accompanied by hyperactivity. CONCLUSIONS These experiments revealed that vPAG α1ARs increase arousal, promote glutamatergic input onto vPAGDA neurons, and are abundantly expressed on astrocytes. Activation of locus coeruleus inputs, vPAG astrocytes, or vPAGDA neurons increase sleep latency but do not produce hyperactivity. Together, these results support an arousal circuit whereby noradrenergic transmission at astrocytic α1ARs activates wake-promoting vPAGDA neurons via glutamate transmission.

Fgf13 Identified as a Novel Cause of GEFS

Commentary Genetic epilepsy with febrile seizures plus (GEFS+) is a dominantly inherited disorder that is characterized by febrile seizures that sometimes persist beyond 6 years of age and the development of epilepsy. Mutations in at least 6 genes have been shown to cause GEFS+, and additional loci have been mapped, indicating that more genes are yet to be identified. Among the known GEFS+ genes, mutations in the voltage-gated sodium channel SCN1A are the most frequently observed, accounting for about 10% of cases (1). In a recent study by Puranam and colleagues, which is the focus of this commentary, fibroblast growth factor 13 (FGF13) was identified as a new GEFS+ gene. The proband of the small pedigree reported in this study was a 21-year-old male who experienced a simple febrile seizure at the age of 6 and subsequently developed temporal lobe seizures and cognitive deficits. His 19-year-old sibling experienced 4 febrile seizures between 1.5 to 2 years of age and developed afebrile seizures at age 4 yet appears to be seizure-free now. Interestingly, the underlying genetic defect was determined to be a disruption of FGF13 (on the X chromosome) due to a balanced translocation between the X chromosome and chromosome 14. This translocation was inherited from the mother who had a history of febrile seizures as an infant. Consistent with expression on the X chromosome, male Fgf13 knock-out mice die at embryonic day 12.5, while heterozygous female mice exhibit a normal lifespan and 50% reduction in Fgf13 expression. Consistent with clinical presentation, mutant mice have spontaneous seizures and are susceptible to hyperthermia-induced seizures (a model of febrile seizure susceptibility). Fgf13 expression was observed in both excitatory and inhibitory neurons of the adult mouse hippocampus, and hippocampal slices from Fgf13 knockout mice exhibited decreased inhibition and increased excitation when compared to wild-type mice. Fibroblast growth factors (FGFs) comprise a family of polypeptides that are highly conserved in both amino acid sequence and gene structure and are involved in a broad range of cellular activities. FGF13, along with FGF11, FGF12, and FGF14, belong to a subset of FGFs termed “fibroblast growth factor homologous factors” (FHFs) that are primarily expressed in the nervous system. Several lines of evidence support a mechanistic overlap between Fgf13 and SCN1A-associated epilepsy. Firstly, FGF11–FGF14 are involved in the regulation of sodium channel function; and FGF13 has been shown to bind directly to the C-terminus of SCN1A, thereby affecting channel trafficking and modulation of channel activity (2). Secondly, as demonstrated in this manuscript, hippocampal slices from Fgf13 knockout mice exhibit decreased inhibitory and increased excitatory synaptic inputs. Similar alterations in neuronal excitability have been observed in Scn1a mutants, suggesting that reduced excitability of inhibitory interneurons is likely a consequence of mutations in both genes. In addition, neuronal cultures from rat embryonic cortex exhibit increased GAD-immunopositive neurons when treated with Fgf13 (3), suggesting that reduced Fgf13 expression would lead to decreased GABA levels. While the authors convincingly demonstrate that FGF13 is the causal gene in this GEFS+ pedigree, several questions remain: First, the frequency of FGF13 mutations in GEFS+ is Disruption of Fgf13 Causes Synaptic Excitatory–Inhibitory Imbalance and Genetic Epilepsy and Febrile Seizures Plus.