Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons.

Expressions of voltage-gated sodium channels Nav1.1 and Nav1.2, encoded by SCN1A and SCN2A genes, respectively, have been reported to be mutually exclusive in most brain regions. In juvenile and adult neocortex, Nav1.1 is predominantly expressed in inhibitory neurons while Nav1.2 is in excitatory neurons. Although a distinct subpopulation of layer V (L5) neocortical excitatory neurons were also reported to express Nav1.1, their nature has been uncharacterized. In hippocampus, Nav1.1 has been proposed to be expressed only in inhibitory neurons. By using newly generated transgenic mouse lines expressing Scn1a promoter-driven green fluorescent protein (GFP), here we confirm the mutually exclusive expressions of Nav1.1 and Nav1.2 and the absence of Nav1.1 in hippocampal excitatory neurons. We also show that Nav1.1 is expressed in inhibitory and a subpopulation of excitatory neurons not only in L5 but all layers of neocortex. By using neocortical excitatory projection neuron markers including FEZF2 for L5 pyramidal tract (PT) and TBR1 for layer VI (L6) cortico-thalamic (CT) projection neurons, we further show that most L5 PT neurons and a minor subpopulation of layer II/III (L2/3) cortico-cortical (CC) neurons express Nav1.1 while the majority of L6 CT, L5/6 cortico-striatal (CS), and L2/3 CC neurons express Nav1.2. These observations now contribute to the elucidation of pathological neural circuits for diseases such as epilepsies and neurodevelopmental disorders caused by SCN1A and SCN2A mutations.

A recurrent PJA1 variant in trigonocephaly and neurodevelopmental disorders.

OBJECTIVE: Neurodevelopmental disorders (NDDs) often associate with epilepsy or craniofacial malformations. Recent large-scale DNA analyses identified hundreds of candidate genes for NDDs, but a large portion of the cases still remain unexplained. We aimed to identify novel candidate genes for NDDs. METHODS: We performed exome sequencing of 95 patients with NDDs including 51 with trigonocephaly and subsequent targeted sequencing of additional 463 NDD patients, functional analyses of variant in vitro, and evaluations of autism spectrum disorder (ASD)-like phenotypes and seizure-related phenotypes in vivo. RESULTS: We identified de novo truncation variants in nine novel genes; CYP1A1, C14orf119, FLI1, CYB5R4, SEL1L2, RAB11FIP2, ZMYND8, ZNF143, and MSX2. MSX2 variants have been described in patients with cranial malformations, and our present patient with the MSX2 de novo truncation variant showed cranial meningocele and partial epilepsy. MSX2 protein is known to be ubiquitinated by an E3 ubiquitin ligase PJA1, and interestingly we found a PJA1 hemizygous p.Arg376Cys variant recurrently in seven Japanese NDD patients; five with trigonocephaly and one with partial epilepsy, and the variant was absent in 886 Japanese control individuals. Pja1 knock-in mice carrying p.Arg365Cys, which is equivalent to p.Arg376Cys in human, showed a significant decrease in PJA1 protein amount, suggesting a loss-of-function effect of the variant. Pja1 knockout mice displayed moderate deficits in isolation-induced ultrasonic vocalizations and increased seizure susceptibility to pentylenetetrazole. INTERPRETATION: These findings propose novel candidate genes including PJA1 and MSX2 for NDDs associated with craniofacial abnormalities and/or epilepsy.

Scn2a haploinsufficient mice display a spectrum of phenotypes affecting anxiety, sociability, memory flexibility and ampakine CX516 rescues their hyperactivity.

Background: Mutations of the SCN2A gene encoding a voltage-gated sodium channel alpha-II subunit Nav1.2 are associated with neurological disorders such as epilepsy, autism spectrum disorders, intellectual disability, and schizophrenia. However, causal relationships and pathogenic mechanisms underlying these neurological defects, especially social and psychiatric features, remain to be elucidated. Methods: We investigated the behavior of mice with a conventional or conditional deletion of Scn2a in a comprehensive test battery including open field, elevated plus maze, light-dark box, three chambers, social dominance tube, resident-intruder, ultrasonic vocalization, and fear conditioning tests. We further monitored the effects of the positive allosteric modulator of AMPA receptors CX516 on these model mice. Results: Conventional heterozygous Scn2a knockout mice (Scn2aKO/+) displayed novelty-induced exploratory hyperactivity and increased rearing. The increased vertical activity was reproduced by heterozygous inactivation of Scn2a in dorsal-telencephalic excitatory neurons but not in inhibitory neurons. Moreover, these phenotypes were rescued by treating Scn2aKO/+ mice with CX516. Additionally, Scn2aKO/+ mice displayed mild social behavior impairment, enhanced fear conditioning, and deficient fear extinction. Neuronal activity was intensified in the medial prefrontal cortex of Scn2aKO/+ mice, with an increase in the gamma band. Conclusions: Scn2aKO/+ mice exhibit a spectrum of phenotypes commonly observed in models of schizophrenia and autism spectrum disorder. Treatment with the CX516 ampakine, which ameliorates hyperactivity in these mice, could be a potential therapeutic strategy to rescue some of the disease phenotypes.

Impaired cortico-striatal excitatory transmission triggers epilepsy.

STXBP1 and SCN2A gene mutations are observed in patients with epilepsies, although the circuit basis remains elusive. Here, we show that mice with haplodeficiency for these genes exhibit absence seizures with spike-and-wave discharges (SWDs) initiated by reduced cortical excitatory transmission into the striatum. Mice deficient for Stxbp1 or Scn2a in cortico-striatal but not cortico-thalamic neurons reproduce SWDs. In Stxbp1 haplodeficient mice, there is a reduction in excitatory transmission from the neocortex to striatal fast-spiking interneurons (FSIs). FSI activity transiently decreases at SWD onset, and pharmacological potentiation of AMPA receptors in the striatum but not in the thalamus suppresses SWDs. Furthermore, in wild-type mice, pharmacological inhibition of cortico-striatal FSI excitatory transmission triggers absence and convulsive seizures in a dose-dependent manner. These findings suggest that impaired cortico-striatal excitatory transmission is a plausible mechanism that triggers epilepsy in Stxbp1 and Scn2a haplodeficient mice.

Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice.

Mutations in the SCN2A gene encoding a voltage-gated sodium channel Nav1.2 are associated with epilepsies, intellectual disability, and autism. SCN2A gain-of-function mutations cause early-onset severe epilepsies, while loss-of-function mutations cause autism with milder and/or later-onset epilepsies. Here we show that both heterozygous Scn2a-knockout and knock-in mice harboring a patient-derived nonsense mutation exhibit ethosuximide-sensitive absence-like seizures associated with spike-and-wave discharges at adult stages. Unexpectedly, identical seizures are reproduced and even more prominent in mice with heterozygous Scn2a deletion specifically in dorsal-telencephalic (e.g., neocortical and hippocampal) excitatory neurons, but are undetected in mice with selective Scn2a deletion in inhibitory neurons. In adult cerebral cortex of wild-type mice, most Nav1.2 is expressed in excitatory neurons with a steady increase and redistribution from proximal (i.e., axon initial segments) to distal axons. These results indicate a pivotal role of Nav1.2 haplodeficiency in excitatory neurons in epilepsies of patients with SCN2A loss-of-function mutations.

[18F]fluorodeoxyglucose-positron emission tomography study of genetically confirmed patients with Dravet syndrome.

OBJECTIVE: To understand cerebral brain dysfunction in patients with Dravet syndrome (DS), we conducted a [18F]fluorodeoxyglucose-positron emission tomography (FDG-PET) study in patients with DS whose SCN1A gene variant was confirmed. METHODS: FDG-PET was performed on eight patients with DS. A SCN1A mutation analysis revealed missense variants in four patients and truncation variants in four patients. The patients' ages at the time of the PET study were 2, 2, 2, 3, 6, 13, 20, and 29 years old, respectively. The patients' developmental/intelligence quotient at the time of the PET study were 62, 52, 64, 35, 30, 15, and <25, respectively. The mean standardized uptake value (SUV) was calculated in four segments (frontal, temporal, parietal, and occipital) for the semi-quantitative analysis of 18F-FDG uptake. This value represents the average of the regions of interest in each lobe and was divided by the average SUV of the cerebellar hemisphere of each patient and compared between the patients with DS and the diseased controls. RESULTS: Glucose uptake in patients with DS decreased significantly, particularly in those ≥6 years old. Importantly, a comparison between the younger and older patients with DS revealed that glucose uptake was normal in patients who were ≤3 years (2, 2, 2, and 3 years), whereas a profound reduction in glucose uptake in the fronto-temporo-parietal-occipital cortices was observed in patients ≥ 6 years (6, 13, 20, and 29 years). Magnetic resonance imaging revealed no detectable atrophic legions or other changes in the cerebral cortices of patients ≥ 6 years of age. SIGNIFICANCE: The present study showed a remarkable reduction in cerebral glucose metabolism in multiple lobes for the first time, which became obvious after the late infantile period. These findings may indicate a functional neuroimaging aspect of epileptic encephalopathy of DS or a feature of the SCN1A variant itself.

Impairments in social novelty recognition and spatial memory in mice with conditional deletion of Scn1a in parvalbumin-expressing cells.

Loss of function mutations in the SCN1A gene, which encodes the voltage-gated sodium channel Nav1.1, have been described in the majority of Dravet syndrome patients presenting with epileptic seizures, hyperactivity, autistic traits, and cognitive decline. We previously reported predominant Nav1.1 expression in parvalbumin-expressing (PV+) inhibitory neurons in juvenile mouse brain and observed epileptic seizures in mice with selective deletion of Scn1a in PV+ cells mediated by PV-Cre transgene expression (Scn1afl/+/PV-Cre-TG). Here we investigate the behavior of Scn1afl/+/PV-Cre-TG mice using a comprehensive battery of behavioral tests. We observed that Scn1afl/+/PV-Cre-TG mice display hyperactive behavior, impaired social novelty recognition, and altered spatial memory. We also generated Scn1afl/+/SST-Cre-KI mice with a selective Scn1a deletion in somatostatin-expressing (SST+) inhibitory neurons using an SST-IRES-Cre knock-in driver line. We observed that Scn1afl/+/SST-Cre-KI mice display no spontaneous convulsive seizures and that Scn1afl/+/SST-Cre-KI mice have a lowered threshold temperature for hyperthermia-induced seizures, although their threshold values are much higher than those of Scn1afl/+/PV-Cre-TG mice. We finally show that Scn1afl/+/SST-Cre-KI mice exhibited no noticeable behavioral abnormalities. These observations suggest that impaired Nav1.1 function in PV+ interneurons is critically involved in the pathogenesis of hyperactivity, autistic traits, and cognitive decline, as well as epileptic seizures, in Dravet syndrome.

Potentiation of excitatory synaptic transmission ameliorates aggression in mice with Stxbp1 haploinsufficiency.

Genetic studies point to a major role of de novo mutations in neurodevelopmental disorders of intellectual disability, autism spectrum disorders, and epileptic encephalopathy. The STXBP1 gene encodes the syntaxin-binding protein 1 (Munc18-1) that critically controls synaptic vesicle exocytosis and synaptic transmission. This gene harbors a high frequency of de novo mutations, which may play roles in these neurodevelopmental disorders. However, the system and behavioral-level pathophysiological changes caused by these genetic defects remain poorly understood. Constitutional (Stxbp1+/-), dorsal-telencephalic excitatory (Stxbp1fl/+/Emx), or global inhibitory neuron-specific (Stxbp1fl/+/Vgat) mice were subjected to a behavioral test battery examining locomotor activity, anxiety, fear learning, and social interactions including aggression. Furthermore, measurements of local field potentials in multiple regions of the brain were performed. Stxbp1+/- male mice exhibited enhanced aggressiveness and impaired fear learning associated with elevated gamma activity in several regions of the brain including the prefrontal cortex. Stxbp1fl/+/Emx mice showed fear-learning deficits, but neither Stxbp1fl/+/Emx nor Stxbp1fl/+/Vgat mice showed increased aggressiveness. Pharmacological potentiation of the excitatory transmission at active synapses via the systemic administration of ampakine CX516, which enhances the excitatory postsynaptic function, ameliorated the aggressive phenotype of Stxbp1+/- mice. These findings suggest that synaptic impairments of the dorsal telencephalic and subcortical excitatory neurons cause learning deficits and enhanced aggression in Stxbp1+/- mice, respectively. Additionally, normalizing the excitatory synaptic transmission is a potential therapeutic option for managing aggressiveness in patients with STXBP1 mutations.

Nav1.2 is expressed in caudal ganglionic eminence-derived disinhibitory interneurons: Mutually exclusive distributions of Nav1.1 and Nav1.2.

Nav1.1 and Nav1.2 are the voltage-gated sodium channel pore-forming alpha I and II subunits, encoded by the genes SCN1A and SCN2A. Although mutations of both genes have similarly been described in patients with epilepsy, autism and/or intellectual disability, their expression sites in brain are largely distinct. Nav1.1 was shown to be expressed dominantly in parvalbumin (PV)-positive or somatostatin (SST)-positive inhibitory neurons and in a sparsely-distributed subpopulation of excitatory neurons. In contrast, Nav1.2 has been reported to be dominantly expressed in excitatory neurons. Here we show that Nav1.2 is also expressed in caudal ganglionic eminence (CGE)-derived inhibitory neurons, and expressions of Nav1.1 and Nav1.2 are mutually-exclusive in many of brain regions including neocortex, hippocampus, cerebellum, striatum and globus pallidus. In neocortex at postnatal day 15, in addition to the expression in excitatory neurons we show that Nav1.2 is expressed in reelin (RLN)-positive/SST-negative inhibitory neurons that are presumably single-bouquet cells because of their cortical layer I-limited distribution, and vasoactive intestinal peptide (VIP)-positive neurons that would be multipolar cell because of their layer I/II margin and layer VI distribution. Although Nav1.2 has previously been reported to be expressed in SST-positive cells, we here show that Nav1.2 is not expressed in either of PV-positive or SST-positive inhibitory neurons. PV-positive and SST-positive inhibitory neurons derive from medial ganglionic eminence (MGE) and innervate excitatory neurons, while VIP-positive and RLN-positive/SST-negative inhibitory neurons derive from CGE, innervate on inhibitory neurons and play disinhibitory roles in the neural network. Our results therefore indicate that, while Nav1.1 is expressed in MEG-derived inhibitory neurons, Nav1.2 is expressed in CGE-derived disinhibitory interneurons in addition to excitatory neurons. These findings should contribute to understanding of the pathology of neurodevelopmental diseases caused by SCN2A mutations.

Nav1.1 haploinsufficiency in excitatory neurons ameliorates seizure-associated sudden death in a mouse model of Dravet syndrome.

Dravet syndrome is a severe epileptic encephalopathy mainly caused by heterozygous mutations in the SCN1A gene encoding a voltage-gated sodium channel Nav1.1. We previously reported dense localization of Nav1.1 in parvalbumin (PV)-positive inhibitory interneurons in mice and abnormal firing of those neurons in Nav1.1-deficient mice. In the present study, we investigated the physiologic consequence of selective Nav1.1 deletion in mouse global inhibitory neurons, forebrain excitatory neurons or PV cells, using vesicular GABA transporter (VGAT)-Cre, empty spiracles homolog 1 (Emx1)-Cre or PV-Cre recombinase drivers. We show that selective Nav1.1 deletion using VGAT-Cre causes epileptic seizures and premature death that are unexpectedly more severe than those observed in constitutive Nav1.1-deficient mice. Nav1.1 deletion using Emx1-Cre does not cause any noticeable abnormalities in mice; however, the severe lethality observed with VGAT-Cre-driven Nav1.1 deletion is rescued by additional Nav1.1 deletion using Emx1-Cre. In addition to predominant expression in PV interneurons, we detected Nav1.1 in subpopulations of excitatory neurons, including entorhino-hippocampal projection neurons, a subpopulation of neocortical layer V excitatory neurons, and thalamo-cortical projection neurons. We further show that even minimal selective Nav1.1 deletion, using PV-Cre, is sufficient to cause spontaneous epileptic seizures and ataxia in mice. Overall, our results indicate that functional impairment of PV inhibitory neurons with Nav1.1 haploinsufficiency contributes to the epileptic pathology of Dravet syndrome, and show for the first time that Nav1.1 haploinsufficiency in excitatory neurons has an ameliorating effect on the pathology.

A homozygous mutation of voltage-gated sodium channel ß(I) gene SCN1B in a patient with Dravet syndrome.

Dravet syndrome is a severe form of epileptic encephalopathy characterized by early onset epileptic seizures followed by ataxia and cognitive decline. Approximately 80% of patients with Dravet syndrome have been associated with heterozygous mutations in SCN1A gene encoding voltage-gated sodium channel (VGSC) α(I) subunit, whereas a homozygous mutation (p.Arg125Cys) of SCN1B gene encoding VGSC ß(I) subunit was recently described in a patient with Dravet syndrome. To further examine the involvement of homozygous SCN1B mutations in the etiology of Dravet syndrome, we performed mutational analyses on SCN1B in 286 patients with epileptic disorders, including 67 patients with Dravet syndrome who have been negative for SCN1A and SCN2A mutations. In the cohort, we found one additional homozygous mutation (p.Ile106Phe) in a patient with Dravet syndrome. The identified homozygous SCN1B mutations indicate that SCN1B is an etiologic candidate underlying Dravet syndrome.

Deletions of SCN1A 5' genomic region with promoter activity in Dravet syndrome.

Mutations involving the voltage-gated sodium channel alpha(I) gene SCN1A are major genetic causes of childhood epileptic disorders, as typified by Dravet syndrome. Here we investigated the upstream regions of the SCN1A 5' noncoding exons and found two major regions with promoter activity. These two major promoters were simultaneously active in various brain regions and in most neurons. Using multiplex ligation-dependent probe amplification (MLPA) assays with probes for the 5' noncoding exons, their upstream regions, and all coding exons of SCN1A, we investigated 130 epileptic patients who did not show any SCN1A mutations by sequence analysis of all coding exons and exon-intron boundaries. Among 71 Dravet syndrome patients, we found two patients with heterozygous microdeletions removing the 5' noncoding exons and regions with promoter activity but not affecting the coding exons. We also identified four patients with deletions/duplication in the coding region. One patient with symptomatic focal epilepsy also showed a deletion in the coding region. This study provides the first case of microdeletion limited to the SCN1A 5' promoter region with the coding sequence preserved, and indicates the critical involvement of this upstream region in the molecular pathology of Dravet syndrome.

Acute encephalopathy with a truncation mutation in the SCN1A gene: a case report.

A girl aged 1 year 9 months had recurrent episodes of febrile status epilepticus. She recovered completely after the first three episodes. However, at 9 months she developed acute encephalopathy resulting in severe neurologic sequelae. Diffusion-weighted magnetic resonance imaging revealed diffuse high-intensity signals over the cortex and subcortical white matter in the acute phase and severe diffuse cerebral atrophy in the chronic phase. Mutations were detected in the neuronal voltage-gated sodium channel alpha subunit type 1 (SCN1A) gene. SCN1A sequence analysis revealed a truncation mutation:e x1-c.126Adel (D43fs). Our patient was likely afflicted by severe myoclonic epilepsy in infancy, and the fourth episode of status epilepticus was similar to acute encephalopathy. This report provides further insight into the molecular pathophysiology underlying acute encephalopathy.

Nav1.1 localizes to axons of parvalbumin-positive inhibitory interneurons: a circuit basis for epileptic seizures in mice carrying an Scn1a gene mutation.

Loss-of-function mutations in human SCN1A gene encoding Nav1.1 are associated with a severe epileptic disorder known as severe myoclonic epilepsy in infancy. Here, we generated and characterized a knock-in mouse line with a loss-of-function nonsense mutation in the Scn1a gene. Both homozygous and heterozygous knock-in mice developed epileptic seizures within the first postnatal month. Immunohistochemical analyses revealed that, in the developing neocortex, Nav1.1 was clustered predominantly at the axon initial segments of parvalbumin-positive (PV) interneurons. In heterozygous knock-in mice, trains of evoked action potentials in these fast-spiking, inhibitory cells exhibited pronounced spike amplitude decrement late in the burst. Our data indicate that Nav1.1 plays critical roles in the spike output from PV interneurons and, furthermore, that the specifically altered function of these inhibitory circuits may contribute to epileptic seizures in the mice.

Patients with a sodium channel alpha 1 gene mutation show wide phenotypic variation.

We investigated the roles of mutations in voltage-gated sodium channel alpha 1 subunit gene (SCN1A) in epilepsies and psychiatric disorders. The SCN1A gene was screened for mutations in three unrelated Japanese families with generalized epilepsy with febrile seizure plus (GEFS+), febrile seizure with myoclonic seizures, or intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC). In the family with GEFS+, one individual was affected with panic disorder and seizures, and another individual was diagnosed with Asperger syndrome and seizures. The novel mutation V1366I was found in all probands and patients with psychiatric disorders of the three families. These results suggest that SCN1A mutations may confer susceptibility to psychiatric disorders in addition to variable epileptic seizures. Unidentified modifiers may play critical roles in determining the ultimate phenotype of patients with sodium channel mutations.

SCN1A mutation mosaicism in a family with severe myoclonic epilepsy in infancy.

PURPOSE: To investigate the genetic background of familial severe myoclonic epilepsy in infancy (SMEI) cases. METHODS: We performed mutation analyses of the sodium-channel gene SCN1A in two Japanese brothers with clinical features of SMEI and their parents, who had no history of febrile and epileptic seizures. RESULTS: Each patient showed nucleotide changes (c.[730G>T; 735G>T; 736A>T]) in the coding exon 6 of SCN1A that led to a truncation of the channel protein. Their father showed no mutations, but their mother showed the same mutation in a subpopulation of lymphocytes. CONCLUSIONS: The maternal mosaicism explains the identical SCN1A mutations in the two brothers. This highlights the importance of investigating parental mosaicism even in sporadic SMEI cases.

A Kv4.2 truncation mutation in a patient with temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) has a multifactorial etiology involving developmental, environmental, and genetic components. Here, we report a voltage-gated potassium channel gene mutation found in a TLE patient, namely a Kv4.2 truncation mutation. Kv4.2 channels, encoded by the KCND2 gene, mediate A currents in the brain. The identified mutation corresponds to an N587fsX1 amino acid change, predicted to produce a truncated Kv4.2 protein lacking the last 44 amino acids in the carboxyl terminal. Electrophysiological analysis indicates attenuated K+ current density in cells expressing this Kv4.2-N587fsX1 mutant channel, which is consistent with a model of aberrant neuronal excitability characteristic of TLE. Our observations, together with other lines of evidence, raise the intriguing possibility of a role for KCND2 in the etiology of TLE.

A nonsense mutation of the sodium channel gene SCN2A in a patient with intractable epilepsy and mental decline.

Mutations, exclusively missense, of voltage-gated sodium channel alpha subunit type 1 (SCN1A) and type 2 (SCN2A) genes were reported in patients with idiopathic epilepsy: generalized epilepsy with febrile seizures plus. Nonsense and frameshift mutations of SCN1A, by contrast, were identified in intractable epilepsy: severe myoclonic epilepsy in infancy (SMEI). Here we describe a first nonsense mutation of SCN2A in a patient with intractable epilepsy and severe mental decline. The phenotype is similar to SMEI but distinct because of partial epilepsy, delayed onset (1 year 7 months), and absence of temperature sensitivity. A mutational analysis revealed that the patient had a heterozygous de novo nonsense mutation R102X of SCN2A. Patch-clamp analysis of Na(v)1.2 wild-type channels and the R102X mutant protein coexpressed in human embryonic kidney 293 cells showed that the truncated mutant protein shifted the voltage dependence of inactivation of wild-type channels in the hyperpolarizing direction. Analysis of the subcellular localization of R102X truncated protein suggested that its dominant negative effect could arise from direct or indirect cytoskeletal interactions of the mutant protein. Haploinsufficiency of Na(v)1.2 protein is one plausible explanation for the pathology of this patient; however, our biophysical findings suggest that the R102X truncated protein exerts a dominant negative effect leading to the patient's intractable epilepsy.