Scn1a gene reactivation after symptom onset rescues pathological phenotypes in a mouse model of Dravet syndrome.

Dravet syndrome is a severe epileptic encephalopathy caused primarily by haploinsufficiency of the SCN1A gene. Repetitive seizures can lead to endurable and untreatable neurological deficits. Whether this severe pathology is reversible after symptom onset remains unknown. To address this question, we generated a Scn1a conditional knock-in mouse model (Scn1a Stop/+) in which Scn1a expression can be re-activated on-demand during the mouse lifetime. Scn1a gene disruption leads to the development of seizures, often associated with sudden unexpected death in epilepsy (SUDEP) and behavioral alterations including hyperactivity, social interaction deficits and cognitive impairment starting from the second/third week of age. However, we showed that Scn1a gene re-activation when symptoms were already manifested (P30) led to a complete rescue of both spontaneous and thermic inducible seizures, marked amelioration of behavioral abnormalities and normalization of hippocampal fast-spiking interneuron firing. We also identified dramatic gene expression alterations, including those associated with astrogliosis in Dravet syndrome mice, that, accordingly, were rescued by Scn1a gene expression normalization at P30. Interestingly, regaining of Nav1.1 physiological level rescued seizures also in adult Dravet syndrome mice (P90) after months of repetitive attacks. Overall, these findings represent a solid proof-of-concept highlighting that disease phenotype reversibility can be achieved when Scn1a gene activity is efficiently reconstituted in brain cells.

Modeling NaV1.1/SCN1A sodium channel mutations in a microcircuit with realistic ion concentration dynamics suggests differential GABAergic mechanisms leading to hyperexcitability in epilepsy and hemiplegic migraine.

Loss of function mutations of SCN1A, the gene coding for the voltage-gated sodium channel NaV1.1, cause different types of epilepsy, whereas gain of function mutations cause sporadic and familial hemiplegic migraine type 3 (FHM-3). However, it is not clear yet how these opposite effects can induce paroxysmal pathological activities involving neuronal networks' hyperexcitability that are specific of epilepsy (seizures) or migraine (cortical spreading depolarization, CSD). To better understand differential mechanisms leading to the initiation of these pathological activities, we used a two-neuron conductance-based model of interconnected GABAergic and pyramidal glutamatergic neurons, in which we incorporated ionic concentration dynamics in both neurons. We modeled FHM-3 mutations by increasing the persistent sodium current in the interneuron and epileptogenic mutations by decreasing the sodium conductance in the interneuron. Therefore, we studied both FHM-3 and epileptogenic mutations within the same framework, modifying only two parameters. In our model, the key effect of gain of function FHM-3 mutations is ion fluxes modification at each action potential (in particular the larger activation of voltage-gated potassium channels induced by the NaV1.1 gain of function), and the resulting CSD-triggering extracellular potassium accumulation, which is not caused only by modifications of firing frequency. Loss of function epileptogenic mutations, on the other hand, increase GABAergic neurons' susceptibility to depolarization block, without major modifications of firing frequency before it. Our modeling results connect qualitatively to experimental data: potassium accumulation in the case of FHM-3 mutations and facilitated depolarization block of the GABAergic neuron in the case of epileptogenic mutations. Both these effects can lead to pyramidal neuron hyperexcitability, inducing in the migraine condition depolarization block of both the GABAergic and the pyramidal neuron. Overall, our findings suggest different mechanisms of network hyperexcitability for migraine and epileptogenic NaV1.1 mutations, implying that the modifications of firing frequency may not be the only relevant pathological mechanism.

Dravet syndrome and Dravet syndrome-like phenotype: a systematic review of the SCN1A and PCDH19 variants.

Dravet syndrome (DS) is a rare and severe epileptic syndrome of childhood with prevalence between 1/22,000 and 1/49,900 of live births. Approximately 80% of patients with this syndrome present SCN1A pathogenic variants, which encodes an alpha subunit of a neural voltage-dependent sodium channel. There is a correlation between PCDH19 pathogenic variants, encodes the protocadherin 19, and a similar disease to DS known as DS-like phenotype. The present review aims to clarify the differences between DS and DS-like phenotype according to the SCN1A and PCDH19 variants. A systematic review was conducted in PubMed and Virtual Health Library (VHL) databases, using "Dravet Syndrome" and "Severe Myoclonic Epilepsy in Infancy (SMEI)" search words, selecting cohort of studies published in journal with impact factor of two or greater. The systematic review was according to the Preferred Reporting Items for Systematic Review and Meta-Analysis recommendations. Nineteen studies were included in the present review, and a significant proportion of patients with DS-carrying SCN1A was greater than patients with DS-like phenotype-harboring PCDH19 variants (76.6% versus 23.4%). When clinical and genetic data were correlated, autism was predominantly observed in patients with DS-like-carrying PCDH19 variants compared to SCN1A variant carriers (62.5% versus 37.5%, respectively, P-value = 0.044, P-value corrected = 0.198). In addition, it was noticed a significant predisposition to hyperthermia during epilepsy crisis in individuals carrying PCDH19 variants (P-value = 0.003; P-value corrected = 0.027). The present review is the first to point out differences between the DS and DS-like phenotype according to the SCN1A and PCDH19 variants.

Disordered autonomic function during exposure to moderate heat or exercise in a mouse model of Dravet syndrome.

OBJECTIVE: To examine autonomic regulation of core body temperature, heart rate (HR), and breathing rate (BR) in response to moderately elevated ambient temperature or moderate physical exercise in a mouse model of Dravet syndrome (DS). METHODS: We studied video-EEG, ECG, respiration, and temperature in mice with global heterozygous Scn1a knockout (KO) (DS mice), interneuron specific Scn1a KO, and wildtype (WT) mice during exposure to increased environmental temperature and moderate treadmill exercise. RESULTS: Core body temperatures of WT and DS mice were similar during baseline. After 15 mins of heat exposure, the peak value was lower in DS than WT mice. In the following mins of heat exposure, the temperature slowly returned close to baseline level in WT, whereas it remained elevated in DS mice. KO of Scn1a in GABAergic neurons caused similar thermoregulatory deficits in mice. During exercise, the HR increase was less prominent in DS than WT mice. After exercise, the HR was significantly more suppressed in DS. The heart rate variability (HRV) was lower in DS than WT mice during baseline and higher in DS during exercise-recovery periods. SIGNIFICANCE: We found novel abnormalities that expand the spectrum of interictal, ictal, and postictal autonomic dysregulation in DS mice. During mild heat stress, there was a significantly blunted correction of body temperature, and a less suppression of both HR and respiration rate in DS than WT mice. These effects were seen in mice with selective KO of Scn1A in GABAergic neurons. During exercise stress, there was diminished increase in HR, followed by an exaggerated HR suppression and HRV elevation during recovery in DS mice compared to controls. These findings suggest that different environmental stressors can uncover distinct autonomic disturbances in DS mice. Interneurons play an important role in thermoregulation. Understanding the spectrum and mechanisms of autonomic disorders in DS may help develop more effective strategies to prevent seizures and SUDEP.

Delayed maturation of GABAergic signaling in the Scn1a and Scn1b mouse models of Dravet Syndrome.

Dravet syndrome (DS) is a catastrophic developmental and epileptic encephalopathy characterized by severe, pharmacoresistant seizures and the highest risk of Sudden Unexpected Death in Epilepsy (SUDEP) of all epilepsy syndromes. Here, we investigated the time course of maturation of neuronal GABAergic signaling in the Scn1b-/- and Scn1a+/- mouse models of DS. We found that GABAergic signaling remains immature in both DS models, with a depolarized reversal potential for GABAA-evoked currents compared to wildtype in the third postnatal week. Treatment of Scn1b-/- mice with bumetanide resulted in a delay in SUDEP onset compared to controls in a subset of mice, without prevention of seizure activity or amelioration of failure to thrive. We propose that delayed maturation of GABAergic signaling may contribute to epileptogenesis in SCN1B- and SCN1A-linked DS. Thus, targeting the polarity of GABAergic signaling in brain may be an effective therapeutic strategy to reduce SUDEP risk in DS.

Drug repurposing for Dravet syndrome in scn1Lab-/- mutant zebrafish.

Dravet syndrome (DS) is a severe genetic epileptic encephalopathy with onset during the first year of life. Zebrafish models recapitulating human diseases are often used as drug discovery platforms, but also for drug repurposing testing. It was recently shown that pharmacological modulation of three serotonergic (5-HT) receptors (5-HT1D , 5-HT2C , 5-HT2A ) exerts antiseizure effects in a zebrafish scn1Lab-/- mutant model of DS. Using the zebrafish DS model, our aim was to examine the possibility of repurposing efavirenz (EFA), lisuride (LIS), and rizatriptan (RIZA), marketed medicines with a 5-HT on- or off-target profile, as antiepileptic drugs for DS. To examine whether these compounds have a broader antiseizure profile, they were tested in pentylenetetrazol and ethyl ketopentenoate (EKP) zebrafish models. Pharmacological effects were assessed by locomotor behavior, local field potential brain recordings, and bioluminescence. EFA was active in all models, whereas LIS was selectively active in the zebrafish DS model. Mainly, a poor response was observed to RIZA. Taken together, our preclinical results show that LIS could be a potential candidate for DS treatment. EFA was also active in the EKP model, characterized by a high level of treatment resistance, and hence these data are potentially important for future treatment of drug-resistant epilepsy.

RNA-seq Analysis of the SCN1A-KO Model based on CRISPR/Cas9 Genome Editing Technology.

Dravet syndrome (DS) is a disease that is primarily caused by the inactivation of the SCN1A-encoded voltage-gated sodium channel alpha subunit (Nav1.1). In this study, we constructed an SCN1A gene knockout model using CRISPR/Cas9 genome editing technology to deprive the Nav1.1 function in vitro. With mRNA-seq analysis we found abundant gene changes after SCN1A knockout, which associated with various signaling pathways, such as cancer pathways, the PI3K-AKT signaling pathway, the MAPK signaling pathway, and pathways involved in HTLV-I infection. We also noticed changes in the spliceosome, decreased glycolytic capacity, disturbances in calcium signaling pathways, and changes in the potassium, sodium, chloride, and calcium plasma channels after SCN1A knockout. In this study, we have been the first time to discover these changes and summarize them here and hope it would provide some clue for the study of Nav1.1 in the nervous system.

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.

Heat induced temperature dysregulation and seizures in Dravet Syndrome/GEFS+ Gabrg2+/Q390X mice.

It has been established that febrile seizures and its extended syndromes like generalized epilepsy with febrile seizures (FS) plus (GEFS+) and Dravet syndrome have been associated with mutations especially in SCN1A and GABRG2 genes. In patients, the onset of FS is likely due to the combined effect of temperature and inflammation in genetically vulnerable individuals because fever is often associated with infection. Much effort has been spent to understand the mechanisms underlying fever induction of seizures. In addition to the role of cytokines in FS, previous studies in Scn1a+/- knockout mice, a model of Dravet syndrome, indicated that temperature elevation alone could result in seizure generation, and the effect of elevated temperature inducing seizures was age-dependent. Here, we report the thermal effect in a different mouse model of Dravet syndrome, the Gabrg2+/Q390X knockin mouse. We demonstrated age-dependent dysregulated temperature control and that temperature elevation produced myoclonic jerks, generalized tonic clonic seizures (GTCSs) and heightened anxiety-like symptoms in Gabrg2+/Q390X mice. The study indicated that regardless of other inflammatory factors, brief heat alone increased brain excitability and induced multiple types of seizures in Gabrg2+/Q390X mice, suggesting that mutations like GABRG2(Q390X) may alter brain thermal regulation and precipitate seizures during temperature elevations.

A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients.

Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons.

Cognitive Deficits Associated with Nav1.1 Alterations: Involvement of Neuronal Firing Dynamics and Oscillations.

Brain oscillations play a critical role in information processing and may, therefore, be essential to uncovering the mechanisms of cognitive impairment in neurological disease. In Dravet syndrome (DS), a mutation in SCN1A, coding for the voltage-gated sodium channel Nav1.1, is associated with severe cognitive impairment and seizures. While seizure frequency and severity do not correlate with the extent of impairment, the slowing of brain rhythms may be involved. Here we investigate the role of Nav1.1 on brain rhythms and cognition using RNA interference. We demonstrate that knockdown of Nav1.1 impairs fast- and burst-firing properties of neurons in the medial septum in vivo. The proportion of neurons that fired phase-locked to hippocampal theta oscillations was reduced, and medial septal regulation of theta rhythm was disrupted. During a working memory task, this deficit was characterized by a decrease in theta frequency and was negatively correlated with performance. These findings suggest a fundamental role for Nav1.1 in facilitating fast-firing properties in neurons, highlight the importance of precise temporal control of theta frequency for working memory, and imply that Nav1.1 deficits may disrupt information processing in DS via a dysregulation of brain rhythms.

Strain- and age-dependent hippocampal neuron sodium currents correlate with epilepsy severity in Dravet syndrome mice.

Heterozygous loss-of-function SCN1A mutations cause Dravet syndrome, an epileptic encephalopathy of infancy that exhibits variable clinical severity. We utilized a heterozygous Scn1a knockout (Scn1a(+/-)) mouse model of Dravet syndrome to investigate the basis for phenotype variability. These animals exhibit strain-dependent seizure severity and survival. Scn1a(+/-) mice on strain 129S6/SvEvTac (129.Scn1a(+/-)) have no overt phenotype and normal survival compared with Scn1a(+/-) mice bred to C57BL/6J (F1.Scn1a(+/-)) that have severe epilepsy and premature lethality. We tested the hypothesis that strain differences in sodium current (INa) density in hippocampal neurons contribute to these divergent phenotypes. Whole-cell voltage-clamp recording was performed on acutely-dissociated hippocampal neurons from postnatal days 21-24 (P21-24) 129.Scn1a(+/-) or F1.Scn1a(+/-) mice and wild-type littermates. INa density was lower in GABAergic interneurons from F1.Scn1a(+/-) mice compared to wild-type littermates, while on the 129 strain there was no difference in GABAergic interneuron INa density between 129.Scn1a(+/-) mice and wild-type littermate controls. By contrast, INa density was elevated in pyramidal neurons from both 129.Scn1a(+/-) and F1.Scn1a(+/-) mice, and was correlated with more frequent spontaneous action potential firing in these neurons, as well as more sustained firing in F1.Scn1a(+/-) neurons. We also observed age-dependent differences in pyramidal neuron INa density between wild-type and Scn1a(+/-) animals. We conclude that preserved INa density in GABAergic interneurons contributes to the milder phenotype of 129.Scn1a(+/-) mice. Furthermore, elevated INa density in excitatory pyramidal neurons at P21-24 correlates with age-dependent onset of lethality in F1.Scn1a(+/-) mice. Our findings illustrate differences in hippocampal neurons that may underlie strain- and age-dependent phenotype severity in a Dravet syndrome mouse model, and emphasize a contribution of pyramidal neuron excitability.

Hippocampal hyperexcitability and specific epileptiform activity in a mouse model of Dravet syndrome.

PURPOSE: Dravet syndrome (DS) is caused by dominant mutations of the SCN1A gene, encoding the NaV 1.1 sodium channel α subunit. Gene targeted mouse models of DS mutations replicate patients' phenotype and show reduced γ-aminobutyric acid (GABA)ergic inhibition. However, little is known on the properties of network hyperexcitability and on properties of seizure generation in these models. In fact, seizures have been studied thus far with surface electroencephalography (EEG), which did not show if specific brain regions are particularly involved. We have investigated hyperexcitability and epileptiform activities generated in neuronal networks of a mouse model of DS. METHODS: We have studied heterozygous NaV 1.1 knock-out mice performing field potential recordings in combined hippocampal/cortical slices in vitro and video/depth electrode intracerebral recordings in vivo during hyperthermia-induced seizures. KEY FINDINGS: In slices, we have disclosed specific signs of hyperexcitability of hippocampal circuits in both the pre-epileptic and epileptic periods, and a specific epileptiform activity was generated in the hippocampus upon application of the convulsant 4-aminopyridine in the epileptic period. During in vivo hyperthermia-induced seizures, we have observed selective hippocampal activity in early preictal phases and pronounced hippocampal activity in the ictal phase. SIGNIFICANCE: We have identified specific epileptiform activities and signs of network hyperexcitability, and disclosed the important role of the hippocampus in seizure generation in this model. These activities may be potentially used as targets for screenings of antiepileptic approaches.

Mouse with Nav1.1 haploinsufficiency, a model for Dravet syndrome, exhibits lowered sociability and learning impairment.

Dravet syndrome is an intractable epileptic encephalopathy characterized by early onset epileptic seizures followed by cognitive decline, hyperactivity, autistic behaviors and ataxia. Most Dravet syndrome patients possess heterozygous mutations of SCN1A gene encoding voltage-gated sodium channel αI subunit (Nav1.1). We have previously reported that mice heterozygous for a nonsense mutation in Scn1a developed early onset epileptic seizures. However, the learning ability and sociability of the mice remained to be investigated. In the present study, we subjected heterozygous Scn1a mice to a comprehensive behavioral test battery. We found that while heterozygous Scn1a mice had lowered spontaneous motor activity in home cage, they were hyperactive in novel environments. Moreover, the mice had low sociability and poor spatial learning ability that correspond to the autistic behaviors and cognitive decline seen in Dravet syndrome patients. These results suggest that Nav1.1 haploinsufficiency intrinsically contributes to not only epileptic seizures but also lowered sociability and learning impairment in heterozygous Scn1a mutant mice, as it should also be the case in patients with Dravet syndrome.

Preferential inactivation of Scn1a in parvalbumin interneurons increases seizure susceptibility.

Voltage-gated sodium channels (VGSCs) are essential for the generation and propagation of action potentials in electrically excitable cells. Dominant mutations in SCN1A, which encodes the Nav1.1 VGSC α-subunit, underlie several forms of epilepsy, including Dravet syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). Electrophysiological analyses of DS and GEFS+ mouse models have led to the hypothesis that SCN1A mutations reduce the excitability of inhibitory cortical and hippocampal interneurons. To more directly examine the relative contribution of inhibitory interneurons and excitatory pyramidal cells to SCN1A-derived epilepsy, we first compared the expression of Nav1.1 in inhibitory parvalbumin (PV) interneurons and excitatory neurons from P22 mice using fluorescent immunohistochemistry. In the hippocampus and neocortex, 69% of Nav1.1 immunoreactive neurons were also positive for PV. In contrast, 13% and 5% of Nav1.1 positive cells in the hippocampus and neocortex, respectively, were found to co-localize with excitatory cells identified by CaMK2α immunoreactivity. Next, we reduced the expression of Scn1a in either a subset of interneurons (mainly PV interneurons) or excitatory cells by crossing mice heterozygous for a floxed Scn1a allele to either the Ppp1r2-Cre or EMX1-Cre transgenic lines, respectively. The inactivation of one Scn1a allele in interneurons of the neocortex and hippocampus was sufficient to reduce thresholds to flurothyl- and hyperthermia-induced seizures, whereas thresholds were unaltered following inactivation in excitatory cells. Reduced interneuron Scn1a expression also resulted in the generation of spontaneous seizures. These findings provide direct evidence for an important role of PV interneurons in the pathogenesis of Scn1a-derived epilepsies.

Specific deletion of NaV1.1 sodium channels in inhibitory interneurons causes seizures and premature death in a mouse model of Dravet syndrome.

Heterozygous loss-of-function mutations in the brain sodium channel Na(V)1.1 cause Dravet syndrome (DS), a pharmacoresistant infantile-onset epilepsy syndrome with comorbidities of cognitive impairment and premature death. Previous studies using a mouse model of DS revealed reduced sodium currents and impaired excitability in GABAergic interneurons in the hippocampus, leading to the hypothesis that impaired excitability of GABAergic inhibitory neurons is the cause of epilepsy and premature death in DS. However, other classes of GABAergic interneurons are less impaired, so the direct cause of hyperexcitability, epilepsy, and premature death has remained unresolved. We generated a floxed Scn1a mouse line and used the Cre-Lox method driven by an enhancer from the Dlx1,2 locus for conditional deletion of Scn1a in forebrain GABAergic neurons. Immunocytochemical studies demonstrated selective loss of Na(V)1.1 channels in GABAergic interneurons in cerebral cortex and hippocampus. Mice with this deletion died prematurely following generalized tonic-clonic seizures, and they were equally susceptible to thermal induction of seizures as mice with global deletion of Scn1a. Evidently, loss of Na(V)1.1 channels in forebrain GABAergic neurons is both necessary and sufficient to cause epilepsy and premature death in DS.