Neurodevelopmental deficits and cell-type-specific transcriptomic perturbations in a mouse model of HNRNPU haploinsufficiency.

Heterozygous de novo loss-of-function mutations in the gene expression regulator HNRNPU cause an early-onset developmental and epileptic encephalopathy. To gain insight into pathological mechanisms and lay the potential groundwork for developing targeted therapies, we characterized the neurophysiologic and cell-type-specific transcriptomic consequences of a mouse model of HNRNPU haploinsufficiency. Heterozygous mutants demonstrated global developmental delay, impaired ultrasonic vocalizations, cognitive dysfunction and increased seizure susceptibility, thus modeling aspects of the human disease. Single-cell RNA-sequencing of hippocampal and neocortical cells revealed widespread, yet modest, dysregulation of gene expression across mutant neuronal subtypes. We observed an increased burden of differentially-expressed genes in mutant excitatory neurons of the subiculum-a region of the hippocampus implicated in temporal lobe epilepsy. Evaluation of transcriptomic signature reversal as a therapeutic strategy highlights the potential importance of generating cell-type-specific signatures. Overall, this work provides insight into HNRNPU-mediated disease mechanisms and provides a framework for using single-cell RNA-sequencing to study transcriptional regulators implicated in disease.

Single unit analysis and wide-field imaging reveal alterations in excitatory and inhibitory neurons in glioma.

While several studies have attributed the development of tumour-associated seizures to an excitatory-inhibitory imbalance, we have yet to resolve the spatiotemporal interplay between different types of neuron in glioma-infiltrated cortex. Herein, we combined methods for single unit analysis of microelectrode array recordings with wide-field optical mapping of Thy1-GCaMP pyramidal cells in an ex vivo acute slice model of diffusely infiltrating glioma. This enabled simultaneous tracking of individual neurons from both excitatory and inhibitory populations throughout seizure-like events. Moreover, our approach allowed for observation of how the crosstalk between these neurons varied spatially, as we recorded across an extended region of glioma-infiltrated cortex. In tumour-bearing slices, we observed marked alterations in single units classified as putative fast-spiking interneurons, including reduced firing, activity concentrated within excitatory bursts and deficits in local inhibition. These results were correlated with increases in overall excitability. Mechanistic perturbation of this system with the mTOR inhibitor AZD8055 revealed increased firing of putative fast-spiking interneurons and restoration of local inhibition, with concomitant decreases in overall excitability. Altogether, our findings suggest that diffusely infiltrating glioma affect the interplay between excitatory and inhibitory neuronal populations in a reversible manner, highlighting a prominent role for functional mechanisms linked to mTOR activation.

KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy. Amino acids are a key input to this system, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RAGA, and GATOR2, a positive regulator of unknown molecular function. Here we identify a protein complex (KICSTOR) that is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and that is required for amino acid or glucose deprivation to inhibit mTORC1 in cultured human cells. In mice that lack SZT2, mTORC1 signalling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds and recruits GATOR1, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Notably, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signalling. Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signalling, which, like GATOR1, is mutated in human disease.

Overlaps, gaps, and complexities of mouse models of Developmental and Epileptic Encephalopathy.

Mouse models have made innumerable contributions to understanding the genetic basis of neurological disease and pathogenic mechanisms and to therapy development. Here we consider the current state of mouse genetic models of Developmental and Epileptic Encephalopathy (DEE), representing a set of rare but devastating and largely intractable childhood epilepsies. By examining the range of mouse lines available in this rapidly moving field and by detailing both expected and unusual features in representative examples, we highlight lessons learned in an effort to maximize the full potential of this powerful resource for preclinical studies.

Modelling and treating GRIN2A developmental and epileptic encephalopathy in mice.

NMDA receptors play crucial roles in excitatory synaptic transmission. Rare variants in GRIN2A encoding the GluN2A subunit are associated with a spectrum of disorders, ranging from mild speech and language delay to intractable neurodevelopmental disorders, including but not limited to developmental and epileptic encephalopathy. A de novo missense variant, p.Ser644Gly, was identified in a child with this disorder, and Grin2a knock-in mice were generated to model and extend understanding of this intractable childhood disease. Homozygous and heterozygous mutant mice exhibited altered hippocampal morphology at 2 weeks of age, and all homozygotes exhibited lethal tonic-clonic seizures by mid-third week. Heterozygous adults displayed susceptibility to induced generalized seizures, hyperactivity, repetitive and reduced anxiety behaviours, plus several unexpected features, including significant resistance to electrically-induced limbic seizures and to pentylenetetrazole induced tonic-clonic seizures. Multielectrode recordings of neuronal networks revealed hyperexcitability and altered bursting and synchronicity. In heterologous cells, mutant receptors had enhanced NMDA receptor agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. NMDA receptor-mediated synaptic currents in heterozygous hippocampal slices also showed a prolonged deactivation time course. Standard anti-epileptic drug monotherapy was ineffective in the patient. Introduction of NMDA receptor antagonists was correlated with a decrease in seizure burden. Chronic treatment of homozygous mouse pups with NMDA receptor antagonists significantly delayed the onset of lethal seizures but did not prevent them. These studies illustrate the power of using multiple experimental modalities to model and test therapies for severe neurodevelopmental disorders, while revealing significant biological complexities associated with GRIN2A developmental and epileptic encephalopathy.

RNAi-Based Gene Therapy Rescues Developmental and Epileptic Encephalopathy in a Genetic Mouse Model.

Developmental and epileptic encephalopathy (DEE) associated with de novo variants in the gene encoding dynamin-1 (DNM1) is a severe debilitating disease with no pharmacological remedy. Like most genetic DEEs, the majority of DNM1 patients suffer from therapy-resistant seizures and comorbidities such as intellectual disability, developmental delay, and hypotonia. We tested RNAi gene therapy in the Dnm1 fitful mouse model of DEE using a Dnm1-targeted therapeutic microRNA delivered by a self-complementary adeno-associated virus vector. Untreated or control-injected fitful mice have growth delay, severe ataxia, and lethal tonic-clonic seizures by 3 weeks of age. These major impairments are mitigated following a single treatment in newborn mice, along with key underlying cellular features including gliosis, cell death, and aberrant neuronal metabolic activity typically associated with recurrent seizures. Our results underscore the potential for RNAi gene therapy to treat DNM1 disease and other genetic DEEs where treatment would require inhibition of the pathogenic gene product.

Altered excitatory transmission onto hippocampal interneurons in the IQSEC2 mouse model of X-linked neurodevelopmental disease.

Mutations in the X-linked gene IQSEC2 are associated with multiple cases of epilepsy, epileptic encephalopathy, intellectual disability and autism spectrum disorder, the mechanistic understanding and successful treatment of which remain a significant challenge in IQSEC2 and related neurodevelopmental genetic diseases. To investigate disease etiology, we studied behaviors and synaptic function in IQSEC2 deficient mice. Hemizygous Iqsec2 null males exhibit growth deficits, hyperambulation and hyperanxiety phenotypes. Adult hemizygotes experience lethal spontaneous seizures, but paradoxically have a significantly increased threshold to electrically induced limbic seizures and relative resistance to chemically induced seizures. Although there are no gross defects in brain morphology, hemizygotes exhibit stark hippocampal reactive astrogliosis. Electrophysiological recordings of hippocampal neurons reveal increased excitatory drive specifically onto interneurons, and significant alterations in intrinsic electrical properties specific to the interneuron population. As they age, hemizygotes also develop an increased abundance of parvalbumin-positive interneurons in the hippocampus, neurons in which IQSEC2 is expressed in addition to the excitatory neurons. These findings point to a novel role of IQSEC2 in hippocampal interneuron synaptic function and development with implications for a class of intractable neurodevelopmental diseases.

Arfgef1 haploinsufficiency in mice alters neuronal endosome composition and decreases membrane surface postsynaptic GABAA receptors.

ARFGEF1 encodes a guanine exchange factor involved in intracellular vesicle trafficking, and is a candidate gene for childhood genetic epilepsies. To model ARFGEF1 haploinsufficiency observed in a recent Lennox Gastaut Syndrome patient, we studied a frameshift mutation (Arfgef1fs) in mice. Arfgef1fs/+ pups exhibit signs of developmental delay, and Arfgef1fs/+ adults have a significantly decreased threshold to induced seizures but do not experience spontaneous seizures. Histologically, the Arfgef1fs/+ brain exhibits a disruption in the apical lining of the dentate gyrus and altered spine morphology of deep layer neurons. In primary hippocampal neuron culture, dendritic surface and synaptic but not total GABAA receptors (GABAAR) are reduced in Arfgef1fs/+ neurons with an accompanying decrease in the number of GABAAR-containing recycling endosomes in cell body. Arfgef1fs/+ neurons also display differences in the relative ratio of Arf6+:Rab11+:TrfR+ recycling endosomes. Although the GABAAR-containing early endosomes in Arfgef1fs/+ neurons are comparable to wildtype, Arfgef1fs/+ neurons show an increase in the number of GABAAR-containing lysosomes in dendrite and cell body. Together, the altered endosome composition and decreased neuronal surface GABAAR results suggests a mechanism whereby impaired neuronal inhibition leads to seizure susceptibility.

PRAS: Predicting functional targets of RNA binding proteins based on CLIP-seq peaks.

RNA-protein interaction plays important roles in post-transcriptional regulation. Recent advancements in cross-linking and immunoprecipitation followed by sequencing (CLIP-seq) technologies make it possible to detect the binding peaks of a given RNA binding protein (RBP) at transcriptome scale. However, it is still challenging to predict the functional consequences of RBP binding peaks. In this study, we propose the Protein-RNA Association Strength (PRAS), which integrates the intensities and positions of the binding peaks of RBPs for functional mRNA targets prediction. We illustrate the superiority of PRAS over existing approaches on predicting the functional targets of two related but divergent CELF (CUGBP, ELAV-like factor) RBPs in mouse brain and muscle. We also demonstrate the potential of PRAS for wide adoption by applying it to the enhanced CLIP-seq (eCLIP) datasets of 37 RNA decay related RBPs in two human cell lines. PRAS can be utilized to investigate any RBPs with available CLIP-seq peaks. PRAS is freely available at http://ouyanglab.jax.org/pras/.

Inhibition of microRNA 128 promotes excitability of cultured cortical neuronal networks.

Cultured neuronal networks monitored with microelectrode arrays (MEAs) have been used widely to evaluate pharmaceutical compounds for potential neurotoxic effects. A newer application of MEAs has been in the development of in vitro models of neurological disease. Here, we directly evaluated the utility of MEAs to recapitulate in vivo phenotypes of mature microRNA-128 (miR-128) deficiency, which causes fatal seizures in mice. We show that inhibition of miR-128 results in significantly increased neuronal activity in cultured neuronal networks derived from primary mouse cortical neurons. These results support the utility of MEAs in developing in vitro models of neuroexcitability disorders, such as epilepsy, and further suggest that MEAs provide an effective tool for the rapid identification of microRNAs that promote seizures when dysregulated.

meaRtools: An R package for the analysis of neuronal networks recorded on microelectrode arrays.

Here we present an open-source R package 'meaRtools' that provides a platform for analyzing neuronal networks recorded on Microelectrode Arrays (MEAs). Cultured neuronal networks monitored with MEAs are now being widely used to characterize in vitro models of neurological disorders and to evaluate pharmaceutical compounds. meaRtools provides core algorithms for MEA spike train analysis, feature extraction, statistical analysis and plotting of multiple MEA recordings with multiple genotypes and treatments. meaRtools functionality covers novel solutions for spike train analysis, including algorithms to assess electrode cross-correlation using the spike train tiling coefficient (STTC), mutual information, synchronized bursts and entropy within cultured wells. Also integrated is a solution to account for bursts variability originating from mixed-cell neuronal cultures. The package provides a statistical platform built specifically for MEA data that can combine multiple MEA recordings and compare extracted features between different genetic models or treatments. We demonstrate the utilization of meaRtools to successfully identify epilepsy-like phenotypes in neuronal networks from Celf4 knockout mice. The package is freely available under the GPL license (GPL> = 3) and is updated frequently on the CRAN web-server repository. The package, along with full documentation can be downloaded from: https://cran.r-project.org/web/packages/meaRtools/.

Independent Neuronal Origin of Seizures and Behavioral Comorbidities in an Animal Model of a Severe Childhood Genetic Epileptic Encephalopathy.

The childhood epileptic encephalopathies (EE's) are seizure disorders that broadly impact development including cognitive, sensory and motor progress with severe consequences and comorbidities. Recently, mutations in DNM1 (dynamin 1) have been implicated in two EE syndromes, Lennox-Gastaut Syndrome and Infantile Spasms. Dnm1 encodes dynamin 1, a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Dnm1Ftfl or "fitful" mice carry a spontaneous mutation in the mouse ortholog of DNM1 and recapitulate many of the disease features associated with human DNM1 patients, providing a relevant disease model of human EE's. In order to examine the cellular etiology of seizures and behavioral and neurological comorbidities, we engineered a conditional Dnm1Ftfl mouse model of DNM1 EE. Observations of Dnm1Ftfl/flox mice in combination with various neuronal subpopulation specific cre strains demonstrate unique seizure phenotypes and clear separation of major neurobehavioral comorbidities from severe seizures associated with the germline model. This demonstration of pleiotropy suggests that treating seizures per se may not prevent severe comorbidity observed in EE associated with dynamin-1 mutations, and is likely to have implications for other genetic forms of EE.

Unraveling genetic modifiers in the gria4 mouse model of absence epilepsy.

Absence epilepsy (AE) is a common type of genetic generalized epilepsy (GGE), particularly in children. AE and GGE are complex genetic diseases with few causal variants identified to date. Gria4 deficient mice provide a model of AE, one for which the common laboratory inbred strain C3H/HeJ (HeJ) harbors a natural IAP retrotransposon insertion in Gria4 that reduces its expression 8-fold. Between C3H and non-seizing strains such as C57BL/6, genetic modifiers alter disease severity. Even C3H substrains have surprising variation in the duration and incidence of spike-wave discharges (SWD), the characteristic electroencephalographic feature of absence seizures. Here we discovered extensive IAP retrotransposition in the C3H substrain, and identified a HeJ-private IAP in the Pcnxl2 gene, which encodes a putative multi-transmembrane protein of unknown function, resulting in decreased expression. By creating new Pcnxl2 frameshift alleles using TALEN mutagenesis, we show that Pcnxl2 deficiency is responsible for mitigating the seizure phenotype - making Pcnxl2 the first known modifier gene for absence seizures in any species. This finding gave us a handle on genetic complexity between strains, directing us to use another C3H substrain to map additional modifiers including validation of a Chr 15 locus that profoundly affects the severity of SWD episodes. Together these new findings expand our knowledge of how natural variation modulates seizures, and highlights the feasibility of characterizing and validating modifiers in mouse strains and substrains in the post-genome sequence era.

Dynamin 1 isoform roles in a mouse model of severe childhood epileptic encephalopathy.

Dynamin 1 is a large neuron-specific GTPase involved in the endocytosis and recycling of pre-synaptic membranes and synaptic vesicles. Mutations in the gene encoding dynamin 1 (DNM1) underlie two epileptic encephalopathy syndromes, Lennox-Gastaut Syndrome and Infantile Spasms. Mice homozygous for the Dnm1 "fitful" mutation, a non-synonymous coding variant in an alternatively spliced exon of Dnm1 (exon 10a; isoform designation: Dnm1a(Ftfl)) have an epileptic encephalopathy-like disorder including lethal early onset seizures, locomotor and neurosensory deficits. Although fitful heterozygotes have milder recurrent seizures later in life, suggesting an additive or semi-dominant mechanism, the molecular etiology must also consider the fact that Dnm1a(Ftfl) exerts a dominant negative effect on endocytosis in vitro. Another complication is that the fitful mutation induces alterations in the relative abundance of Dnm1 splice variants; mutants have a downregulation of Dnm1a and an upregulation of Dnm1b, changes which may contribute to the epileptic pathology. To examine whether Dnm1a loss of function, Dnm1a(Ftfl) dominance or compensation by Dnm1b is the most critical for severe seizures, we studied alternate isoform-specific mutant mice. Mice lacking Dnm1 exon 10a or Dnm1 exon 10b have neither spontaneous seizures nor other overt abnormalities, suggesting that in normal conditions the major role of each isoform is redundant. However, in the presence of Dnm1a(Ftfl) only exon 10a deleted mice experience severe seizures. These results reveal functional differences between Dnm1a and Dnm1b isoforms in the presence of a challenge, i.e. toxic Dnm1(Ftfl), while reinforcing its effect explicitly in this model of severe pediatric epilepsy.

Loss of MeCP2 from forebrain excitatory neurons leads to cortical hyperexcitation and seizures.

Mutations of MECP2 cause Rett syndrome (RTT), a neurodevelopmental disorder leading to loss of motor and cognitive functions, impaired social interactions, and seizure at young ages. Defects of neuronal circuit development and function are thought to be responsible for the symptoms of RTT. The majority of RTT patients show recurrent seizures, indicating that neuronal hyperexcitation is a common feature of RTT. However, mechanisms underlying hyperexcitation in RTT are poorly understood. Here we show that deletion of Mecp2 from cortical excitatory neurons but not forebrain inhibitory neurons in the mouse leads to spontaneous seizures. Selective deletion of Mecp2 from excitatory but not inhibitory neurons in the forebrain reduces GABAergic transmission in layer 5 pyramidal neurons in the prefrontal and somatosensory cortices. Loss of MeCP2 from cortical excitatory neurons reduces the number of GABAergic synapses in the cortex, and enhances the excitability of layer 5 pyramidal neurons. Using single-cell deletion of Mecp2 in layer 2/3 pyramidal neurons, we show that GABAergic transmission is reduced in neurons without MeCP2, but is normal in neighboring neurons with MeCP2. Together, these results suggest that MeCP2 in cortical excitatory neurons plays a critical role in the regulation of GABAergic transmission and cortical excitability.

Modes and regulation of endocytic membrane retrieval in mouse auditory hair cells.

Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1-pHluorin imaging and membrane capacitance (Cm) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1-pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was ∼6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca(2+) influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic Cm decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster Cm decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic Cm decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis.

CELF4 regulates translation and local abundance of a vast set of mRNAs, including genes associated with regulation of synaptic function.

RNA-binding proteins have emerged as causal agents of complex neurological diseases. Mice deficient for neuronal RNA-binding protein CELF4 have a complex neurological disorder with epilepsy as a prominent feature. Human CELF4 has recently been associated with clinical features similar to those seen in mutant mice. CELF4 is expressed primarily in excitatory neurons, including large pyramidal cells of the cerebral cortex and hippocampus, and it regulates excitatory but not inhibitory neurotransmission. We examined mechanisms underlying neuronal hyperexcitability in Celf4 mutants by identifying CELF4 target mRNAs and assessing their fate in the absence of CELF4 in view of their known functions. CELF4 binds to at least 15%-20% of the transcriptome, with striking specificity for the mRNA 3' untranslated region. CELF4 mRNA targets encode a variety of proteins, many of which are well established in neuron development and function. While the overall abundance of these mRNA targets is often dysregulated in Celf4 deficient mice, the actual expression changes are modest at the steady-state level. In contrast, by examining the transcriptome of polysome fractions and the mRNA distribution along the neuronal cell body-neuropil axis, we found that CELF4 is critical for maintaining mRNA stability and availability for translation. Among biological processes associated with CELF4 targets that accumulate in neuropil of mutants, regulation of synaptic plasticity and transmission are the most prominent. Together with a related study of the impact of CELF4 loss on sodium channel Na(v)1.6 function, we suggest that CELF4 deficiency leads to abnormal neuronal function by combining a specific effect on neuronal excitation with a general impairment of synaptic transmission. These results also expand our understanding of the vital roles RNA-binding proteins play in regulating and shaping the activity of neural circuits.

Elevated Id2 expression results in precocious neural stem cell depletion and abnormal brain development.

Id2 is a helix-loop-helix transcription factor essential for normal development, and its expression is dysregulated in many human neurological conditions. Although it is speculated that elevated Id2 levels contribute to the pathogenesis of these disorders, it is unknown whether dysregulated Id2 expression is sufficient to perturb normal brain development or function. Here, we show that mice with elevated Id2 expression during embryonic stages develop microcephaly, and that females in particular are prone to generalized tonic-clonic seizures. Analyses of Id2 transgenic brains indicate that Id2 activity is highly cell context specific: elevated Id2 expression in naive neural stem cells (NSCs) in early neuroepithelium induces apoptosis and loss of NSCs and intermediate progenitors. Activation of Id2 in maturing neuroepithelium results in less severe phenotypes and is accompanied by elevation of G1 cyclin expression and p53 target gene expression. In contrast, activation of Id2 in committed intermediate progenitors has no significant phenotype. Functional analysis with Id2-overexpressing and Id2-null NSCs shows that Id2 negatively regulates NSC self-renewal in vivo, in contrast to previous cell culture experiments. Deletion of p53 function from Id2-transgenic brains rescues apoptosis and results in increased incidence of brain tumors. Furthermore, Id2 overexpression normalizes the increased self-renewal of p53-null NSCs, suggesting that Id2 activates and modulates the p53 pathway in NSCs. Together, these data suggest that elevated Id2 expression in embryonic brains can cause deregulated NSC self-renewal, differentiation, and survival that manifest in multiple neurological outcomes in mature brains, including microcephaly, seizures, and brain tumors.

Antiepileptic activity of preferential inhibitors of persistent sodium current.

OBJECTIVE: Evidence from basic neurophysiology and molecular genetics has implicated persistent sodium current conducted by voltage-gated sodium (NaV ) channels as a contributor to the pathogenesis of epilepsy. Many antiepileptic drugs target NaV channels and modulate neuronal excitability, mainly by a use-dependent block of transient sodium current, although suppression of persistent current may also contribute to the efficacy of these drugs. We hypothesized that a drug or compound capable of preferential inhibition of persistent sodium current would have antiepileptic activity. METHODS: We examined the antiepileptic activity of two selective persistent sodium current blockers ranolazine, a U.S. Food and Drug Administration (FDA)-approved drug for treatment of angina pectoris, and GS967, a novel compound with more potent effects on persistent current, in the epileptic Scn2a(Q54) mouse model. We also examined the effect of GS967 in the maximal electroshock model and evaluated effects of the compound on neuronal excitability, propensity for hilar neuron loss, development of mossy fiber sprouting, and survival of Scn2a(Q54) mice. RESULTS: We found that ranolazine was capable of reducing seizure frequency by approximately 50% in Scn2a(Q54) mice. The more potent persistent current blocker GS967 reduced seizure frequency by >90% in Scn2a(Q54) mice and protected against induced seizures in the maximal electroshock model. GS967 greatly attenuated abnormal spontaneous action potential firing in pyramidal neurons acutely isolated from Scn2a(Q54) mice. In addition to seizure suppression in vivo, GS967 treatment greatly improved the survival of Scn2a(Q54) mice, prevented hilar neuron loss, and suppressed the development of hippocampal mossy fiber sprouting. SIGNIFICANCE: Our findings indicate that the selective persistent sodium current blocker GS967 has potent antiepileptic activity and that this compound could inform development of new agents.

Aberrant sodium channel activity in the complex seizure disorder of Celf4 mutant mice.

Mice deficient for CELF4, a neuronal RNA-binding protein, have a complex seizure disorder that includes both convulsive and non-convulsive seizures, and is dependent upon Celf4 gene dosage and mouse strain background. It was previously shown that Celf4 is expressed predominantly in excitatory neurons, and that deficiency results in abnormal excitatory synaptic neurotransmission. To examine the physiological and molecular basis of this, we studied Celf4-deficient neurons in brain slices. Assessment of intrinsic properties of layer V cortical pyramidal neurons showed that neurons from mutant heterozygotes and homozygotes have a lower action potential (AP) initiation threshold and a larger AP gain when compared with wild-type neurons. Celf4 mutant neurons also demonstrate an increase in persistent sodium current (I(NaP)) and a hyperpolarizing shift in the voltage dependence of activation. As part of a related study, we find that CELF4 directly binds Scn8a mRNA, encoding sodium channel Na(v)1.6, the primary instigator of AP at the axon initial segment (AIS) and the main carrier of I(NaP). In the present study we find that CELF4 deficiency results in a dramatic elevation in the expression of Na(v)1.6 protein at the AIS in both null and heterozygous neurons. Together these results suggest that activation of Na(v)1.6 plays a crucial role in seizure generation in this complex model of neurological disease.