Preictal dysfunctions of inhibitory interneurons paradoxically lead to their rebound hyperactivity and to low-voltage-fast onset seizures in Dravet syndrome.

Epilepsies have numerous specific mechanisms. The understanding of neural dynamics leading to seizures is important for disclosing pathological mechanisms and developing therapeutic approaches. We investigated electrographic activities and neural dynamics leading to convulsive seizures in patients and mouse models of Dravet syndrome (DS), a developmental and epileptic encephalopathy in which hypoexcitability of GABAergic neurons is considered to be the main dysfunction. We analyzed EEGs from DS patients carrying a SCN1A pathogenic variant, as well as epidural electrocorticograms, hippocampal local field potentials, and hippocampal single-unit neuronal activities in Scn1a+/- and Scn1aRH/+ DS mice. Strikingly, most seizures had low-voltage-fast onset in both patients and mice, which is thought to be generated by hyperactivity of GABAergic interneurons, the opposite of the main pathological mechanism of DS. Analyzing single-unit recordings, we observed that temporal disorganization of the firing of putative interneurons in the period immediately before the seizure (preictal) precedes the increase of their activity at seizure onset, together with the entire neuronal network. Moreover, we found early signatures of the preictal period in the spectral features of hippocampal and cortical field potential of Scn1a mice and of patients' EEG, which are consistent with the dysfunctions that we observed in single neurons and that allowed seizure prediction. Therefore, the perturbed preictal activity of interneurons leads to their hyperactivity at the onset of generalized seizures, which have low-voltage-fast features that are similar to those observed in other epilepsies and are triggered by hyperactivity of GABAergic neurons. Preictal spectral features may be used as predictive seizure biomarkers.

Integrating Proteomics and Transcriptomics Reveals the Potential Pathways of Hippocampal Neuron Apoptosis in Dravet Syndrome Model Mice.

An important component contributing to the onset of epilepsy is the death of hippocampal neurons. Several studies have shown that Dravet syndrome model mice: Scn1a KO mice have a high number of apoptotic neurons following seizures, but the precise mechanism underlying this remains unclear. The aim of this research was to elucidate the potential molecular mechanism of neuronal apoptosis in Scn1a KO mice by integrating proteomics and transcriptomics, with the ultimate goal of offering better neuroprotection. We found that apoptotic processes were enriched in both proteomic and transcriptomic GO analyses, and KEGG results also indicated that differential proteins and genes play a role in neurotransmission, the cell cycle, apoptosis, and neuroinflammation. Then, we examined the upstream and downstream KGML interactions of the pathways to determine the relationship between the two omics, and we found that the HIF-1 signaling pathway plays a significant role in the onset and apoptosis of epilepsy. Meanwhile, the expression of the apoptosis-related protein VHL decreased in this pathway, and the expression of p21 was upregulated. Therefore, this study suggests that VHL/HIF-1α/p21 might be involved in the apoptosis of hippocampal neurons in Scn1a KO mice.

Microglia Mitigate Neuronal Activation in a Zebrafish Model of Dravet Syndrome.

It has been known for a long time that epileptic seizures provoke brain neuroinflammation involving the activation of microglial cells. However, the role of these cells in this disease context and the consequences of their inflammatory activation on subsequent neuron network activity remain poorly understood so far. To fill this gap of knowledge and gain a better understanding of the role of microglia in the pathophysiology of epilepsy, we used an established zebrafish Dravet syndrome epilepsy model based on Scn1Lab sodium channel loss-of-function, combined with live microglia and neuronal Ca2+ imaging, local field potential (LFP) recording, and genetic microglia ablation. Data showed that microglial cells in scn1Lab-deficient larvae experiencing epileptiform seizures displayed morphological and biochemical changes characteristic of M1-like pro-inflammatory activation; i.e., reduced branching, amoeboid-like morphology, and marked increase in the number of microglia expressing pro-inflammatory cytokine Il1ß. More importantly, LFP recording, Ca2+ imaging, and swimming behavior analysis showed that microglia-depleted scn1Lab-KD larvae displayed an increase in epileptiform seizure-like neuron activation when compared to that seen in scn1Lab-KD individuals with microglia. These findings strongly suggest that despite microglia activation and the synthesis of pro-inflammatory cytokines, these cells provide neuroprotective activities to epileptic neuronal networks, making these cells a promising therapeutic target in epilepsy.

A high seizure burden increases several prostaglandin species in the hippocampus of a Scn1a+/- mouse model of Dravet syndrome.

Dravet syndrome is an intractable epilepsy with a high seizure burden that is resistant to current anti-seizure medications. There is evidence that neuroinflammation plays a role in epilepsy and seizures, however few studies have specifically examined neuroinflammation in Dravet syndrome under conditions of a higher seizure burden. Here we used an established genetic mouse model of Dravet syndrome (Scn1a+/- mice), to examine whether a higher seizure burden impacts the number and morphology of microglia in the hippocampus. Moreover, we examined whether a high seizure burden influences classical inflammatory mediators in this brain region. Scn1a+/- mice with a high seizure burden induced by thermal priming displayed a localised reduction in microglial cell density in the granule cell layer and subgranular zone of the dentate gyrus, regions important to postnatal neurogenesis. However, microglial cell number and morphology remained unchanged in other hippocampal subfields. The high seizure burden in Scn1a+/- mice did not affect hippocampal mRNA expression of classical inflammatory mediators such as interleukin 1ß and tumour necrosis factor α, but increased cyclooxygenase 2 (COX-2) expression. We then quantified hippocampal levels of prostanoids that arise from COX-2 mediated metabolism of fatty acids and found that Scn1a+/- mice with a high seizure burden displayed increased hippocampal concentrations of numerous prostaglandins, notably PGF2α, PGE2, PGD2, and 6-K-PGF1A, compared to Scn1a+/- mice with a low seizure burden. In conclusion, a high seizure burden increased hippocampal concentrations of various prostaglandin mediators in a mouse model of Dravet syndrome. Future studies could interrogate the prostaglandin pathways to further better understand their role in the pathophysiology of Dravet syndrome.

Ndnf Interneuron Excitability Is Spared in a Mouse Model of Dravet Syndrome.

Dravet syndrome (DS) is a neurodevelopmental disorder characterized by epilepsy, developmental delay/intellectual disability, and features of autism spectrum disorder, caused by heterozygous loss-of-function variants in SCN1A encoding the voltage-gated sodium channel α subunit Nav1.1. The dominant model of DS pathogenesis is the "interneuron hypothesis," whereby GABAergic interneurons (INs) express and preferentially rely on Nav1.1-containing sodium channels for action potential (AP) generation. This has been shown for three of the major subclasses of cerebral cortex GABAergic INs: those expressing parvalbumin (PV), somatostatin, and vasoactive intestinal peptide. Here, we define the function of a fourth major subclass of INs expressing neuron-derived neurotrophic factor (Ndnf) in male and female DS (Scn1a+/-) mice. Patch-clamp electrophysiological recordings of Ndnf-INs in brain slices from Scn1a+/â mice and WT controls reveal normal intrinsic membrane properties, properties of AP generation and repetitive firing, and synaptic transmission across development. Immunohistochemistry shows that Nav1.1 is strongly expressed at the axon initial segment (AIS) of PV-expressing INs but is absent at the Ndnf-IN AIS. In vivo two-photon calcium imaging demonstrates that Ndnf-INs in Scn1a+/â mice are recruited similarly to WT controls during arousal. These results suggest that Ndnf-INs are the only major IN subclass that does not prominently rely on Nav1.1 for AP generation and thus retain their excitability in DS. The discovery of a major IN subclass with preserved function in the Scn1a+/â mouse model adds further complexity to the "interneuron hypothesis" and highlights the importance of considering cell-type heterogeneity when investigating mechanisms underlying neurodevelopmental disorders.

Functional Characteristics of the Nav1.1 p.Arg1596Cys Mutation Associated with Varying Severity of Epilepsy Phenotypes.

Mutations of the SCN1A gene, which encodes the voltage-dependent Na+ channel's α subunit, are associated with diverse epileptic syndromes ranging in severity, even intra-family, from febrile seizures to epileptic encephalopathy. The underlying cause of this variability is unknown, suggesting the involvement of additional factors. The aim of our study was to describe the properties of mutated channels and investigate genetic causes for clinical syndromes' variability in the family of five SCN1A gene p.Arg1596Cys mutation carriers. The analysis of additional genetic factors influencing SCN1A-associated phenotypes was conducted through exome sequencing (WES). To assess the impact of mutations, we used patch clamp analysis of mutated channels expressed in HEK cells and in vivo neural excitability studies (NESs). In cells expressing the mutant channel, sodium currents were reduced. NESs indicated increased excitability of peripheral motor neurons in mutation carriers. WES showed the absence of non-SCA1 pathogenic variants that could be causative of disease in the family. Variants of uncertain significance in three genes, as potential modifiers of the most severe phenotype, were identified. The p.Arg1596Cys substitution inhibits channel function, affecting steady-state inactivation kinetics. Its clinical manifestations involve not only epileptic symptoms but also increased excitability of peripheral motor fibers. The role of Nav1.1 in excitatory neurons cannot be ruled out as a significant factor of the clinical phenotype.

Preferential expression of SCN1A in GABAergic neurons improves survival and epileptic phenotype in a mouse model of Dravet syndrome.

The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease. KEY MESSAGES: Adenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting. An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons. A hybrid promoter allows preferential expression of transgenes in GABAergic neurons. Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.

MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control.

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy.

Viral vector-mediated expression of NaV1.1, after seizure onset, reduces epilepsy in mice with Dravet syndrome.

Dravet syndrome (DS), an intractable childhood epileptic encephalopathy with a high fatality rate, is typically caused by loss-of-function mutations in one allele of SCN1A, which encodes NaV1.1, a 250-kDa voltage-gated sodium channel. In contrast to other epilepsies, pharmaceutical treatment for DS is limited. Here, we demonstrate that viral vector-mediated delivery of a codon-modified SCN1A open reading frame into the brain improves DS comorbidities in juvenile and adolescent DS mice (Scn1aA1783V/WT). Notably, bilateral vector injections into the hippocampus and/or the thalamus of DS mice increased survival, reduced the occurrence of epileptic spikes, provided protection from thermally induced seizures, corrected background electrocorticographic activity and behavioral deficits, and restored hippocampal inhibition. Together, our results provide a proof of concept for the potential of SCN1A delivery as a therapeutic approach for infants and adolescents with DS-associated comorbidities.

Astrocyte Ca2+ signaling is facilitated in Scn1a+/- mouse model of Dravet syndrome.

Dravet syndrome (DS) is an infantile-onset epileptic encephalopathy. More than 80% of DS patients have a heterozygous mutation in SCN1A, which encodes a subunit of the voltage-gated sodium channel, Nav1.1, in neurons. The roles played by astrocytes, the most abundant glial cell type in the brain, have been investigated in the pathogenesis of epilepsy; however, the specific involvement of astrocytes in DS has not been clarified. In this study, we evaluated Ca2+ signaling in astrocytes using genetically modified mice that have a loss-of-function mutation in Scn1a. We found that the slope of spontaneous Ca2+ spiking was increased without a change in amplitude in Scn1a+/- astrocytes. In addition, ATP-induced transient Ca2+ influx and the slope of Ca2+ spiking were also increased in Scn1a+/- astrocytes. These data indicate that perturbed Ca2+ dynamics in astrocytes may be involved in the pathogenesis of DS.

The Generation of Human iPSC Lines from Three Individuals with Dravet Syndrome and Characterization of Neural Differentiation Markers in iPSC-Derived Ventral Forebrain Organoid Model.

Dravet syndrome (DRVT) is a rare form of neurodevelopmental disorder with a high risk of sudden unexpected death in epilepsy (SUDEP), caused mainly (>80% cases) by mutations in the SCN1A gene, coding the Nav1.1 protein (alfa-subunit of voltage-sensitive sodium channel). Mutations in SCN1A are linked to heterogenous epileptic phenotypes of various types, severity, and patient prognosis. Here we generated iPSC lines from fibroblasts obtained from three individuals affected with DRVT carrying distinct mutations in the SCN1A gene (nonsense mutation p.Ser1516*, missense mutation p.Arg1596His, and splicing mutation c.2589+2dupT). The iPSC lines, generated with the non-integrative approach, retained the distinct SCN1A gene mutation of the donor fibroblasts and were characterized by confirming the expression of the pluripotency markers, the three-germ layer differentiation potential, the absence of exogenous vector expression, and a normal karyotype. The generated iPSC lines were used to establish ventral forebrain organoids, the most affected type of neurons in the pathology of DRVT. The DRVT organoid model will provide an additional resource for deciphering the pathology behind Nav1.1 haploinsufficiency and drug screening to remediate the functional deficits associated with the disease.

NaV1.1 is essential for proprioceptive signaling and motor behaviors.

The voltage-gated sodium channel (NaV), NaV1.1, is well-studied in the central nervous system; conversely, its contribution to peripheral sensory neuron function is more enigmatic. Here, we identify a new role for NaV1.1 in mammalian proprioception. RNAscope analysis and in vitro patch-clamp recordings in genetically identified mouse proprioceptors show ubiquitous channel expression and significant contributions to intrinsic excitability. Notably, genetic deletion of NaV1.1 in sensory neurons caused profound and visible motor coordination deficits in conditional knockout mice of both sexes, similar to conditional Piezo2-knockout animals, suggesting that this channel is a major contributor to sensory proprioceptive transmission. Ex vivo muscle afferent recordings from conditional knockout mice found that loss of NaV1.1 leads to inconsistent and unreliable proprioceptor firing characterized by action potential failures during static muscle stretch; conversely, afferent responses to dynamic vibrations were unaffected. This suggests that while a combination of Piezo2 and other NaV isoforms is sufficient to elicit activity in response to transient stimuli, NaV1.1 is required for transmission of receptor potentials generated during sustained muscle stretch. Impressively, recordings from afferents of heterozygous conditional knockout animals were similarly impaired, and heterozygous conditional knockout mice also exhibited motor behavioral deficits. Thus, NaV1.1 haploinsufficiency in sensory neurons impairs both proprioceptor function and motor behaviors. Importantly, human patients harboring NaV1.1 loss-of-function mutations often present with motor delays and ataxia; therefore, our data suggest that sensory neuron dysfunction contributes to the clinical manifestations of neurological disorders in which NaV1.1 function is compromised. Collectively, we present the first evidence that NaV1.1 is essential for mammalian proprioceptive signaling and behaviors.

"Virtual patch clamp analysis" for predicting the functional significance of pathogenic variants in sodium channels.

OBJECTIVE: Opening of voltage-gated sodium channels is crucial for neuronal depolarization. Proper channel opening and influx of Na+ through the ion pore, is dependent upon binding of Na+ ion to a specific amino-acid motif (DEKA) within the pore. In this study we used molecular dynamic simulations, an advanced bioinformatic tool, to research the dysfunction caused by pathogenic variants in SCN1a, SCN2a and SCN8a genes. METHOD: Molecular dynamic simulations were performed in six patients: three patients with Dravet syndrome (p.Gly177Ala,p.Ser259Arg and p.Met1267Ile, SCN1a), two patients with early onset drug resistant epilepsy(p.Ala263Val, SCN2a and p.Ile251Arg, SCN8a), and a patient with autism (p.Thr155Ala, SCN2a). After predicting the 3D-structure of mutated proteins by homology modeling, time dependent molecular dynamic simulations were performed, using the Schrödinger algorithm. The opening of the sodium channel, including the detachment of the sodium ion to the DEKA motif and pore diameter were assessed. Results were compared to the existent patch clamp analysis in four patients, and consistency with clinical phenotype was noted. RESULTS: The Na+ ion remained attached to DEKA filter longer when compared to wild type in the p.Gly177Ala, p.Ser259Arg,SCN1a, and p.Thr155Ala, SCN2a variants, consistent with loss-of-function. In contrast, it detached quicker from DEKA than wild type in the p.Ala263Val,SCN2a variant, consistent with gain-of-function. In the p.Met1267Ile,SCN1a variant, detachment from DEKA was quicker, but pore diameter decreased, suggesting partial loss-of-function. In the p.Leu251Arg,SCN8a variant, the pore remained opened longer when compared to wild type, consistent with a gain-of-function. The molecular dynamic simulation results were consistent with the existing patch-clamp analysis studies, as well as the clinical phenotype. SIGNIFICANCE: Molecular dynamic simulation can be useful in predicting pathogenicity of variants and the disease phenotype, and selecting targeted treatment based on channel dysfunction. Further development of these bioinformatic tools may lead to "virtual patch-clamp analysis".

SCN1A overexpression, associated with a genomic region marked by a risk variant for a common epilepsy, raises seizure susceptibility.

Mesial temporal lobe epilepsy with hippocampal sclerosis and a history of febrile seizures is associated with common variation at rs7587026, located in the promoter region of SCN1A. We sought to explore possible underlying mechanisms. SCN1A expression was analysed in hippocampal biopsy specimens of individuals with mesial temporal lobe epilepsy with hippocampal sclerosis who underwent surgical treatment, and hippocampal neuronal cell loss was quantitatively assessed using immunohistochemistry. In healthy individuals, hippocampal volume was measured using MRI. Analyses were performed stratified by rs7587026 type. To study the functional consequences of increased SCN1A expression, we generated, using transposon-mediated bacterial artificial chromosome transgenesis, a zebrafish line expressing exogenous scn1a, and performed EEG analysis on larval optic tecta at 4 day post-fertilization. Finally, we used an in vitro promoter analysis to study whether the genetic motif containing rs7587026 influences promoter activity. Hippocampal SCN1A expression differed by rs7587026 genotype (Kruskal-Wallis test P = 0.004). Individuals homozygous for the minor allele showed significantly increased expression compared to those homozygous for the major allele (Dunn's test P = 0.003), and to heterozygotes (Dunn's test P = 0.035). No statistically significant differences in hippocampal neuronal cell loss were observed between the three genotypes. Among 597 healthy participants, individuals homozygous for the minor allele at rs7587026 displayed significantly reduced mean hippocampal volume compared to major allele homozygotes (Cohen's D = - 0.28, P = 0.02), and to heterozygotes (Cohen's D = - 0.36, P = 0.009). Compared to wild type, scn1lab-overexpressing zebrafish larvae exhibited more frequent spontaneous seizures [one-way ANOVA F(4,54) = 6.95 (P < 0.001)]. The number of EEG discharges correlated with the level of scn1lab overexpression [one-way ANOVA F(4,15) = 10.75 (P < 0.001]. Finally, we showed that a 50 bp promoter motif containing rs7587026 exerts a strong regulatory role on SCN1A expression, though we could not directly link this to rs7587026 itself. Our results develop the mechanistic link between rs7587026 and mesial temporal lobe epilepsy with hippocampal sclerosis and a history of febrile seizures. Furthermore, we propose that quantitative precision may be important when increasing SCN1A expression in current strategies aiming to treat seizures in conditions involving SCN1A haploinsufficiency, such as Dravet syndrome.

Developmentally regulated impairment of parvalbumin interneuron synaptic transmission in an experimental model of Dravet syndrome.

Dravet syndrome is a neurodevelopmental disorder characterized by epilepsy, intellectual disability, and sudden death due to pathogenic variants in SCN1A with loss of function of the sodium channel subunit Nav1.1. Nav1.1-expressing parvalbumin GABAergic interneurons (PV-INs) from young Scn1a+/- mice show impaired action potential generation. An approach assessing PV-IN function in the same mice at two time points shows impaired spike generation in all Scn1a+/- mice at postnatal days (P) 16-21, whether deceased prior or surviving to P35, with normalization by P35 in surviving mice. However, PV-IN synaptic transmission is dysfunctional in young Scn1a+/- mice that did not survive and in Scn1a+/- mice ≥ P35. Modeling confirms that PV-IN axonal propagation is more sensitive to decreased sodium conductance than spike generation. These results demonstrate dynamic dysfunction in Dravet syndrome: combined abnormalities of PV-IN spike generation and propagation drives early disease severity, while ongoing dysfunction of synaptic transmission contributes to chronic pathology.

Chronic partial TrkB activation reduces seizures and mortality in a mouse model of Dravet syndrome.

Dravet syndrome (DS) is one of the most severe childhood epilepsies, characterized by intractable seizures and comorbidities including cognitive and social dysfunction and high premature mortality. DS is mainly caused by loss-of-function mutations in the Scn1a gene encoding Nav1.1 that is predominantly expressed in inhibitory parvalbumin-containing (PV) interneurons. Decreased Nav1.1 impairs PV cell function, contributing to DS phenotypes. Effective pharmacological therapy that targets defective PV interneurons is not available. The known role of brain-derived neurotrophic factor (BDNF) in the development and maintenance of interneurons, together with our previous results showing improved PV interneuronal function and antiepileptogenic effects of a TrkB receptor agonist in a posttraumatic epilepsy model, led to the hypothesis that early treatment with a TrkB receptor agonist might prevent or reduce seizure activity in DS mice. To test this hypothesis, we treated DS mice with LM22A-4 (LM), a partial agonist at the BDNF TrkB receptor, for 7 d starting at postnatal day 13 (P13), before the onset of spontaneous seizures. Results from immunohistochemistry, Western blot, whole-cell patch-clamp recording, and in vivo seizure monitoring showed that LM treatment increased the number of perisomatic PV interneuronal synapses around cortical pyramidal cells in layer V, upregulated Nav1.1 in PV neurons, increased inhibitory synaptic transmission, and decreased seizures and the mortality rate in DS mice. The results suggest that early treatment with a partial TrkB receptor agonist may be a promising therapeutic approach to enhance PV interneuron function and reduce epileptogenesis and premature death in DS.

NPRL2 Inhibition of mTORC1 Controls Sodium Channel Expression and Brain Amino Acid Homeostasis.

Genetic mutations in nitrogen permease regulator-like 2 (NPRL2) are associated with a wide spectrum of familial focal epilepsies, autism, and sudden unexpected death of epileptics (SUDEP), but the mechanisms by which NPRL2 contributes to these effects are not well known. NPRL2 is a requisite subunit of the GAP activity toward Rags 1 (GATOR1) complex, which functions as a negative regulator of mammalian target of rapamycin complex 1 (mTORC1) kinase when intracellular amino acids are low. Here, we show that loss of NPRL2 expression in mouse excitatory glutamatergic neurons causes seizures before death, consistent with SUDEP in humans with epilepsy. Additionally, the absence of NPRL2 expression increases mTORC1-dependent signal transduction and significantly alters amino acid homeostasis in the brain. Loss of NPRL2 reduces dendritic branching and increases the strength of electrically stimulated action potentials (APs) in neurons. The increased AP strength is consistent with elevated expression of epilepsy-linked, voltage-gated sodium channels in the NPRL2-deficient brain. Targeted deletion of NPRL2 in primary neurons increases the expression of sodium channel Scn1A, whereas treatment with the pharmacological mTORC1 inhibitor called rapamycin prevents Scn1A upregulation. These studies demonstrate a novel role of NPRL2 and mTORC1 signaling in the regulation of sodium channels, which can contribute to seizures and early lethality.

Genetic and Functional Differences between Duplicated Zebrafish Genes for Human SCN1A.

There are currently seven different zebrafish strains that model Dravet Syndrome, a severe childhood form of epilepsy. These models are based on a set of duplicated genes, scn1laa and scn1lab, which are the homologs for human SCN1A. Disrupting one of the genes would mimic a heterozygous disease state in humans, as the paralog gene is still present. While this 'disease-state model' is widely accepted, there is also evidence that the function of these genes might not be completely the same. By analyzing the functional domains, we discovered several hotspots in the protein that are not conserved, indicating a functional difference. Based on this, we generated scn1Laa knockout zebrafish and compared their phenotype to scn1lab knockouts. The genetic and functional differences we discovered can have implications for the use of zebrafish as a model for Dravet Syndrome.

Hyperexcitable interneurons trigger cortical spreading depression in an Scn1a migraine model.

Cortical spreading depression (CSD), a wave of depolarization followed by depression of cortical activity, is a pathophysiological process implicated in migraine with aura and various other brain pathologies, such as ischemic stroke and traumatic brain injury. To gain insight into the pathophysiology of CSD, we generated a mouse model for a severe monogenic subtype of migraine with aura, familial hemiplegic migraine type 3 (FHM3). FHM3 is caused by mutations in SCN1A, encoding the voltage-gated Na+ channel NaV1.1 predominantly expressed in inhibitory interneurons. Homozygous Scn1aL1649Q knock-in mice died prematurely, whereas heterozygous mice had a normal lifespan. Heterozygous Scn1aL1649Q knock-in mice compared with WT mice displayed a significantly enhanced susceptibility to CSD. We found L1649Q to cause a gain-of-function effect with an impaired Na+-channel inactivation and increased ramp Na+ currents leading to hyperactivity of fast-spiking inhibitory interneurons. Brain slice recordings using K+-sensitive electrodes revealed an increase in extracellular K+ in the early phase of CSD in heterozygous mice, likely representing the mechanistic link between interneuron hyperactivity and CSD initiation. The neuronal phenotype and premature death of homozygous Scn1aL1649Q knock-in mice was partially rescued by GS967, a blocker of persistent Na+ currents. Collectively, our findings identify interneuron hyperactivity as a mechanism to trigger CSD.

Initiation of migraine-related cortical spreading depolarization by hyperactivity of GABAergic neurons and NaV1.1 channels.

Spreading depolarizations (SDs) are involved in migraine, epilepsy, stroke, traumatic brain injury, and subarachnoid hemorrhage. However, the cellular origin and specific differential mechanisms are not clear. Increased glutamatergic activity is thought to be the key factor for generating cortical spreading depression (CSD), a pathological mechanism of migraine. Here, we show that acute pharmacological activation of NaV1.1 (the main Na+ channel of interneurons) or optogenetic-induced hyperactivity of GABAergic interneurons is sufficient to ignite CSD in the neocortex by spiking-generated extracellular K+ build-up. Neither GABAergic nor glutamatergic synaptic transmission were required for CSD initiation. CSD was not generated in other brain areas, suggesting that this is a neocortex-specific mechanism of CSD initiation. Gain-of-function mutations of NaV1.1 (SCN1A) cause familial hemiplegic migraine type-3 (FHM3), a subtype of migraine with aura, of which CSD is the neurophysiological correlate. Our results provide the mechanism linking NaV1.1 gain of function to CSD generation in FHM3. Thus, we reveal the key role of hyperactivity of GABAergic interneurons in a mechanism of CSD initiation, which is relevant as a pathological mechanism of Nav1.1 FHM3 mutations, and possibly also for other types of migraine and diseases in which SDs are involved.