Antisense oligonucleotides restore excitability, GABA signalling and sodium current density in a Dravet syndrome model.

Dravet syndrome is an intractable developmental and epileptic encephalopathy caused by de novo variants in SCN1A resulting in haploinsufficiency of the voltage-gated sodium channel Nav1.1. We showed previously that administration of the antisense oligonucleotide STK-001, also called ASO-22, generated using targeted augmentation of nuclear gene output technology to prevent inclusion of the nonsense-mediated decay, or poison, exon 20N in human SCN1A, increased productive Scn1a transcript and Nav1.1 expression and reduced the incidence of electrographic seizures and sudden unexpected death in epilepsy in a mouse model of Dravet syndrome. Here, we investigated the mechanism of action of ASO-84, a surrogate for ASO-22 that also targets splicing of SCN1A exon 20N, in Scn1a+/- Dravet syndrome mouse brain. Scn1a +/- Dravet syndrome and wild-type mice received a single intracerebroventricular injection of antisense oligonucleotide or vehicle at postnatal Day 2. We examined the electrophysiological properties of cortical pyramidal neurons and parvalbumin-positive fast-spiking interneurons in brain slices at postnatal Days 21-25 and measured sodium currents in parvalbumin-positive interneurons acutely dissociated from postnatal Day 21-25 brain slices. We show that, in untreated Dravet syndrome mice, intrinsic cortical pyramidal neuron excitability was unchanged while cortical parvalbumin-positive interneurons showed biphasic excitability with initial hyperexcitability followed by hypoexcitability and depolarization block. Dravet syndrome parvalbumin-positive interneuron sodium current density was decreased compared to wild-type. GABAergic signalling to cortical pyramidal neurons was reduced in Dravet syndrome mice, suggesting decreased GABA release from interneurons. ASO-84 treatment restored action potential firing, sodium current density and GABAergic signalling in Dravet syndrome parvalbumin-positive interneurons. Our work suggests that interneuron excitability is selectively affected by ASO-84. This new work provides critical insights into the mechanism of action of this antisense oligonucleotide and supports the potential of antisense oligonucleotide-mediated upregulation of Nav1.1 as a successful strategy to treat Dravet syndrome.

Epilepsy and sudden unexpected death in epilepsy in a mouse model of human SCN1B-linked developmental and epileptic encephalopathy.

Voltage-gated sodium channel ß1 subunits are essential proteins that regulate excitability. They modulate sodium and potassium currents, function as cell adhesion molecules and regulate gene transcription following regulated intramembrane proteolysis. Biallelic pathogenic variants in SCN1B, encoding ß1, are linked to developmental and epileptic encephalopathy 52, with clinical features overlapping Dravet syndrome. A recessive variant, SCN1B-c.265C>T, predicting SCN1B-p.R89C, was homozygous in two children of a non-consanguineous family. One child was diagnosed with Dravet syndrome, while the other had a milder phenotype. We identified an unrelated biallelic SCN1B-c.265C>T patient with a clinically more severe phenotype than Dravet syndrome. We used CRISPR/Cas9 to knock-in SCN1B-p.R89C to the mouse Scn1b locus (Scn1bR89/C89). We then rederived the line on the C57BL/6J background to allow comparisons between Scn1bR89/R89 and Scn1bC89/C89 littermates with Scn1b+/+ and Scn1b-/- mice, which are congenic on C57BL/6J, to determine whether the SCN1B-c.265C>T variant results in loss-of-function. Scn1bC89/C89 mice have normal body weights and ∼20% premature mortality, compared with severely reduced body weight and 100% mortality in Scn1b-/- mice. ß1-p.R89C polypeptides are expressed in brain at comparable levels to wild type. In heterologous cells, ß1-p.R89C localizes to the plasma membrane and undergoes regulated intramembrane proteolysis similar to wild type. Heterologous expression of ß1-p.R89C results in sodium channel α subunit subtype specific effects on sodium current. mRNA abundance of Scn2a, Scn3a, Scn5a and Scn1b was increased in Scn1bC89/C89 somatosensory cortex, with no changes in Scn1a. In contrast, Scn1b-/- mouse somatosensory cortex is haploinsufficient for Scn1a, suggesting an additive mechanism for the severity of the null model via disrupted regulation of another Dravet syndrome gene. Scn1bC89/C89 mice are more susceptible to hyperthermia-induced seizures at post-natal Day 15 compared with Scn1bR89/R89 littermates. EEG recordings detected epileptic discharges in young adult Scn1bC89/C89 mice that coincided with convulsive seizures and myoclonic jerks. We compared seizure frequency and duration in a subset of adult Scn1bC89/C89 mice that had been exposed to hyperthermia at post-natal Day 15 versus a subset that were not hyperthermia exposed. No differences in spontaneous seizures were detected between groups. For both groups, the spontaneous seizure pattern was diurnal, occurring with higher frequency during the dark cycle. This work suggests that the SCN1B-c.265C>T variant does not result in complete loss-of-function. Scn1bC89/C89 mice more accurately model SCN1B-linked variants with incomplete loss-of-function compared with Scn1b-/- mice, which model complete loss-of-function, and thus add to our understanding of disease mechanisms as well as our ability to develop new therapeutic strategies.

Neonatal Scn1b-null mice have sinoatrial node dysfunction, altered atrial structure, and atrial fibrillation.

Loss-of-function (LOF) variants in SCN1B, encoding the voltage-gated sodium channel ß1/ß1B subunits, are linked to neurological and cardiovascular diseases. Scn1b-null mice have spontaneous seizures and ventricular arrhythmias and die by approximately 21 days after birth. ß1/ß1B Subunits play critical roles in regulating the excitability of ventricular cardiomyocytes and maintaining ventricular rhythmicity. However, whether they also regulate atrial excitability is unknown. We used neonatal Scn1b-null mice to model the effects of SCN1B LOF on atrial physiology in pediatric patients. Scn1b deletion resulted in altered expression of genes associated with atrial dysfunction. Scn1b-null hearts had a significant accumulation of atrial collagen, increased susceptibility to pacing induced atrial fibrillation (AF), sinoatrial node (SAN) dysfunction, and increased numbers of cholinergic neurons in ganglia that innervate the SAN. Atropine reduced the incidence of AF in null animals. Action potential duration was prolonged in null atrial myocytes, with increased late sodium current density and reduced L-type calcium current density. Scn1b LOF results in altered atrial structure and AF, demonstrating the critical role played by Scn1b in atrial physiology during early postnatal mouse development. Our results suggest that SCN1B LOF variants may significantly impact the developing pediatric heart.

Sodium channel ß1 subunits participate in regulated intramembrane proteolysis-excitation coupling.

Loss-of-function (LOF) variants in SCN1B, encoding voltage-gated sodium channel ß1 subunits, are linked to human diseases with high risk of sudden death, including developmental and epileptic encephalopathy and cardiac arrhythmia. ß1 Subunits modulate the cell-surface localization, gating, and kinetics of sodium channel pore-forming α subunits. They also participate in cell-cell and cell-matrix adhesion, resulting in intracellular signal transduction, promotion of cell migration, calcium handling, and regulation of cell morphology. Here, we investigated regulated intramembrane proteolysis (RIP) of ß1 by BACE1 and γ-secretase and show that ß1 subunits are substrates for sequential RIP by BACE1 and γ-secretase, resulting in the generation of a soluble intracellular domain (ICD) that is translocated to the nucleus. Using RNA sequencing, we identified a subset of genes that are downregulated by ß1-ICD overexpression in heterologous cells but upregulated in Scn1b-null cardiac tissue, which lacks ß1-ICD signaling, suggesting that the ß1-ICD may normally function as a molecular brake on gene transcription in vivo. We propose that human disease variants resulting in SCN1B LOF cause transcriptional dysregulation that contributes to altered excitability. Moreover, these results provide important insights into the mechanism of SCN1B-linked channelopathies, adding RIP-excitation coupling to the multifunctionality of sodium channel ß1 subunits.

Excitatory and inhibitory neuron defects in a mouse model of Scn1b-linked EIEE52.

OBJECTIVE: Human variants in voltage-gated sodium channel (VGSC) α and ß subunit genes are linked to developmental and epileptic encephalopathies (DEEs). Inherited, biallelic, loss-of-function variants in SCN1B, encoding the ß1/ß1B subunits, are linked to early infantile DEE (EIEE52). De novo, monoallelic variants in SCN1A (Nav1.1), SCN2A (Nav1.2), SCN3A (Nav1.3), and SCN8A (Nav1.6) are also linked to DEEs. While these VGSC-linked DEEs have similar presentations, they have diverse mechanisms of altered neuronal excitability. Mouse models have suggested that Scn2a-, Scn3a-, and Scn8a-linked DEE variants are, in general, gain of function, resulting in increased persistent or resurgent sodium current (INa ) and pyramidal neuron hyperexcitability. In contrast, Scn1a-linked DEE variants, in general, are loss-of-function, resulting in decreased INa and hypoexcitability of fast-spiking interneurons. VGSC ß1 subunits associate with Nav1.1, Nav1.2, Nav1.3, and Nav1.6 and are expressed throughout the brain, raising the possibility that insults to both pyramidal and interneuron excitability may drive EIEE52 pathophysiology. METHODS: We investigated excitability defects in pyramidal and parvalbumin-positive (PV +) interneurons in the Scn1b-/- model of EIEE52. We also used Scn1bFL/FL mice to delete Scn1b in specific neuronal populations. RESULTS: Scn1b-/- cortical PV + interneurons were hypoexcitable, with reduced INa density. Scn1b-/- cortical pyramidal neurons had population-specific changes in excitability and impaired INa density. Scn1b deletion in PV + neurons resulted in 100% lethality, whereas deletion in Emx1 + or Camk2a + neurons did not affect survival. INTERPRETATION: This work suggests that SCN1B-linked DEE variants impact both excitatory and inhibitory neurons, leading to the increased severity of EIEE52 relative to other DEEs.

Variant-specific changes in persistent or resurgent sodium current in SCN8A-related epilepsy patient-derived neurons.

Missense variants in the SCN8A voltage-gated sodium channel gene are linked to early-infantile epileptic encephalopathy type 13, also known as SCN8A-related epilepsy. These patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy. The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current properties. To investigate these mechanisms using a patient-specific model, we generated induced pluripotent stem cells from three patients with missense variants in SCN8A: p.R1872>L (Patient 1); p.V1592>L (Patient 2); and p.N1759>S (Patient 3). Using small molecule differentiation into excitatory neurons, induced pluripotent stem cell-derived neurons from all three patients displayed altered sodium currents. Patients 1 and 2 had elevated persistent current, while Patient 3 had increased resurgent current compared to controls. Neurons from all three patients displayed shorter axon initial segment lengths compared to controls. Further analyses focused on one of the patients with increased persistent sodium current (Patient 1) and the patient with increased resurgent current (Patient 3). Excitatory cortical neurons from both patients had prolonged action potential repolarization. Using doxycycline-inducible expression of the neuronal transcription factors neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons, we investigated network activity and response to pharmacotherapies. Both small molecule differentiated and induced patient neurons displayed similar abnormalities in action potential repolarization. Patient induced neurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for SCN8A-related epilepsy patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent sodium currents, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the action potential firing threshold of patient-derived neurons to more depolarized potentials. Two of the patients in this study were prescribed riluzole off-label. Patient 1 had a 50% reduction in seizure frequency. Patient 3 experienced an immediate and dramatic seizure reduction with months of seizure freedom. An additional patient with a SCN8A variant in domain IV of Nav1.6 (p.V1757>I) had a dramatic reduction in seizure frequency for several months after starting riluzole treatment, but then seizures recurred. Our results indicate that patient-specific neurons are useful for modelling SCN8A-related epilepsy and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific neuronal disease modelling offers a useful platform for discovering precision epilepsy therapies.

Sodium channel ß1 subunits are post-translationally modified by tyrosine phosphorylation, S-palmitoylation, and regulated intramembrane proteolysis.

Voltage-gated sodium channel (VGSC) ß1 subunits are multifunctional proteins that modulate the biophysical properties and cell-surface localization of VGSC α subunits and participate in cell-cell and cell-matrix adhesion, all with important implications for intracellular signal transduction, cell migration, and differentiation. Human loss-of-function variants in SCN1B, the gene encoding the VGSC ß1 subunits, are linked to severe diseases with high risk for sudden death, including epileptic encephalopathy and cardiac arrhythmia. We showed previously that ß1 subunits are post-translationally modified by tyrosine phosphorylation. We also showed that ß1 subunits undergo regulated intramembrane proteolysis via the activity of ß-secretase 1 and γ-secretase, resulting in the generation of a soluble intracellular domain, ß1-ICD, which modulates transcription. Here, we report that ß1 subunits are phosphorylated by FYN kinase. Moreover, we show that ß1 subunits are S-palmitoylated. Substitution of a single residue in ß1, Cys-162, to alanine prevented palmitoylation, reduced the level of ß1 polypeptides at the plasma membrane, and reduced the extent of ß1-regulated intramembrane proteolysis, suggesting that the plasma membrane is the site of ß1 proteolytic processing. Treatment with the clathrin-mediated endocytosis inhibitor, Dyngo-4a, re-stored the plasma membrane association of ß1-p.C162A to WT levels. Despite these observations, palmitoylation-null ß1-p.C162A modulated sodium current and sorted to detergent-resistant membrane fractions normally. This is the first demonstration of S-palmitoylation of a VGSC ß subunit, establishing precedence for this post-translational modification as a regulatory mechanism in this protein family.

SCN1B-linked early infantile developmental and epileptic encephalopathy.

OBJECTIVE: Patients with Early Infantile Epileptic Encephalopathy (EIEE) 52 have inherited, homozygous variants in the gene SCN1B, encoding the voltage-gated sodium channel (VGSC) ß1 and ß1B non-pore-forming subunits. METHODS: Here, we describe the detailed electroclinical features of a biallelic SCN1B patient with a previously unreported variant, p.Arg85Cys. RESULTS: The female proband showed hypotonia from birth, multifocal myoclonus at 2.5 months, then focal seizures and myoclonic status epilepticus (SE) at 3 months, triggered by fever. Auditory brainstem response (ABR) showed bilateral hearing loss. Epilepsy was refractory and the patient had virtually no development. Administration of fenfluramine resulted in a significant reduction in seizure frequency and resolution of SE episodes that persisted after a 2-year follow-up. The patient phenotype is more compatible with early infantile developmental and epileptic encephalopathy (DEE) than with typical Dravet syndrome (DS), as previously diagnosed for other patients with homozygous SCN1B variants. Biochemical and electrophysiological analyses of the SCN1B variant expressed in heterologous cells showed cell surface expression of the mutant ß1 subunit, similar to wild-type (WT), but with loss of normal ß1-mediated modification of human Nav 1.1-generated sodium current, suggesting that SCN1B-p.Arg85Cys is a loss-of-function (LOF) variant. INTERPRETATION: Importantly, a review of the literature in light of our results suggests that the term, early infantile developmental and epileptic encephalopathy, is more appropriate than either EIEE or DS to describe biallelic SCN1B patients.

Dravet Syndrome: A Developmental and Epileptic Encephalopathy.

Selective Nav1.1 Activation Rescues Dravet Syndrome Mice From Seizures and Premature Death Richards KL, Milligan CJ, Richardson RJ, Jancovski N, Grunnet M, Jacobson LH, Undheim EAB, Mobli M, Chow CY, Herzig V, Csoti A, Panyi G, Reid CA, King GF, Petrou S. PNAS. 2018;115:E8077-E8085. Dravet syndrome is a catastrophic, pharmaco-resistant epileptic encephalopathy. Disease onset occurs in the first year of life, followed by developmental delay with cognitive and behavioral dysfunction and substantially elevated risk of premature death. The majority of affected individuals harbor a loss-of-function mutation in one allele of SCN1A, which encodes the voltage-gated sodium channel Nav1.1. Brain Nav1.1 is primarily localized to fast-spiking inhibitory interneurons; thus, the mechanism of epileptogenesis in Dravet syndrome is hypothesized to be reduced inhibitory neurotransmission leading to brain hyperexcitability. We show that selective activation of Nav1.1 by venom peptide Hm1a restores the function of inhibitory interneurons from Dravet syndrome mice without affecting the firing of excitatory neurons. Intracerebroventricular infusion of Hm1a rescues Dravet syndrome mice from seizures and premature death. This precision medicine approach, which specifically targets the molecular deficit in Dravet syndrome, presents an opportunity for treatment of this intractable epilepsy. A Transient Developmental Window of Fast-Spiking Interneuron Dysfunction in a Mouse Model of Dravet Syndrome Favero M, Sotuyo NP, Lopez E, Kearney JA, Goldberg EM. J Neurosci. 2018;38:7912-7927. Dravet syndrome is a severe childhood-onset epilepsy largely due to heterozygous loss-of-function mutation of the gene SCN1A, which encodes the type 1 neuronal voltage-gated sodium (Na+) channel α subunit Nav1.1. Prior studies in mouse models of Dravet syndrome ( Scn1a+/- mice) indicate that, in cerebral cortex, Nav1.1 is predominantly expressed in GABAergic interneurons, in particular in parvalbumin-positive fast-spiking basket cell interneurons (PVINs). This has led to a model of Dravet syndrome pathogenesis in which Nav1.1 mutation leads to preferential dysfunction of interneurons, decreased synaptic inhibition, hyperexcitability, and epilepsy. However, such studies have been implemented at early developmental time points. Here, we performed electrophysiological recordings in acute brain slices prepared from male and female Scn1a+/-mice as well as age-matched wild-type littermate controls and found that, later in development, the excitability of PVINs had normalized. Analysis of action potential waveforms indirectly suggests a reorganization of axonal Na+ channels in PVINs from Scn1a+/- mice, a finding supported by immunohistochemical data showing elongation of the axon initial segment. Our results imply that transient impairment of action potential generation by PVINs may contribute to the initial appearance of epilepsy, but is not the mechanism of ongoing, chronic epilepsy in Dravet syndrome. Significance Statement: Dravet syndrome is characterized by normal early development, temperature-sensitive seizures in infancy, progression to treatment-resistant epilepsy, developmental delay, autism, and sudden unexplained death due to mutation in SCN1A encoding the Na+ channel subunit Nav1.1. Prior work has revealed a preferential impact of Nav1.1 loss on the function of GABAergic inhibitory interneurons. However, such data derive exclusively from recordings of neurons in young Scn1a+/- mice. Here, we show that impaired action potential generation observed in parvalbumin-positive fast-spiking interneurons (PVINs) in Scn1a+/- mice during early development has normalized by postnatal day 35. This work suggests that a transient impairment of PVINs contributes to epilepsy onset, but is not the mechanism of ongoing, chronic epilepsy in Dravet syndrome.

Neuronal hyperexcitability in a mouse model of SCN8A epileptic encephalopathy.

Patients with early infantile epileptic encephalopathy (EIEE) experience severe seizures and cognitive impairment and are at increased risk for sudden unexpected death in epilepsy (SUDEP). EIEE13 [Online Mendelian Inheritance in Man (OMIM) # 614558] is caused by de novo missense mutations in the voltage-gated sodium channel gene SCN8A Here, we investigated the neuronal phenotype of a mouse model expressing the gain-of-function SCN8A patient mutation, p.Asn1768Asp (Nav1.6-N1768D). Our results revealed regional and neuronal subtype specificity in the effects of the N1768D mutation. Acutely dissociated hippocampal neurons from Scn8aN1768D/+ mice showed increases in persistent sodium current (INa) density in CA1 pyramidal but not bipolar neurons. In CA3, INa,P was increased in both bipolar and pyramidal neurons. Measurement of action potential (AP) firing in Scn8aN1768D/+ pyramidal neurons in brain slices revealed early afterdepolarization (EAD)-like AP waveforms in CA1 but not in CA3 hippocampal or layer II/III neocortical neurons. The maximum spike frequency evoked by depolarizing current injections in Scn8aN1768D/+ CA1, but not CA3 or neocortical, pyramidal cells was significantly reduced compared with WT. Spontaneous firing was observed in subsets of neurons in CA1 and CA3, but not in the neocortex. The EAD-like waveforms of Scn8aN1768D/+ CA1 hippocampal neurons were blocked by tetrodotoxin, riluzole, and SN-6, implicating elevated persistent INa and reverse mode Na/Ca exchange in the mechanism of hyperexcitability. Our results demonstrate that Scn8a plays a vital role in neuronal excitability and provide insight into the mechanism and future treatment of epileptogenesis in EIEE13.

Scn2b Deletion in Mice Results in Ventricular and Atrial Arrhythmias.

BACKGROUND: Mutations in SCN2B, encoding voltage-gated sodium channel ß2-subunits, are associated with human cardiac arrhythmias, including atrial fibrillation and Brugada syndrome. Because of this, we propose that ß2-subunits play critical roles in the establishment or maintenance of normal cardiac electric activity in vivo. METHODS AND RESULTS: To understand the pathophysiological roles of ß2 in the heart, we investigated the cardiac phenotype of Scn2b null mice. We observed reduced sodium and potassium current densities in ventricular myocytes, as well as conduction slowing in the right ventricular outflow tract region. Functional reentry, resulting from the interplay between slowed conduction, prolonged repolarization, and increased incidence of premature ventricular complexes, was found to underlie the mechanism of spontaneous polymorphic ventricular tachycardia. Scn5a transcript levels were similar in Scn2b null and wild-type ventricles, as were levels of Nav1.5 protein, suggesting that similar to the previous work in neurons, the major function of ß2-subunits in the ventricle is to chaperone voltage-gated sodium channel α-subunits to the plasma membrane. Interestingly, Scn2b deletion resulted in region-specific effects in the heart. Scn2b null atria had normal levels of sodium current density compared with wild type. Scn2b null hearts were more susceptible to atrial fibrillation, had increased levels of fibrosis, and higher repolarization dispersion than wild-type littermates. CONCLUSIONS: Genetic deletion of Scn2b in mice results in ventricular and atrial arrhythmias, consistent with reported SCN2B mutations in human patients.

Cardiac arrhythmia in a mouse model of sodium channel SCN8A epileptic encephalopathy.

Patients with early infantile epileptic encephalopathy (EIEE) are at increased risk for sudden unexpected death in epilepsy (SUDEP). De novo mutations of the sodium channel gene SCN8A, encoding the sodium channel Nav1.6, result in EIEE13 (OMIM 614558), which has a 10% risk of SUDEP. Here, we investigated the cardiac phenotype of a mouse model expressing the gain of function EIEE13 patient mutation p.Asn1768Asp in Scn8a (Nav1.6-N1768D). We tested Scn8aN1768D/+ mice for alterations in cardiac excitability. We observed prolongation of the early stages of action potential (AP) repolarization in mutant myocytes vs. controls. Scn8aN1768D/+ myocytes were hyperexcitable, with a lowered threshold for AP firing, increased incidence of delayed afterdepolarizations, increased calcium transient duration, increased incidence of diastolic calcium release, and ectopic contractility. Calcium transient duration and diastolic calcium release in the mutant myocytes were tetrodotoxin-sensitive. A selective inhibitor of reverse mode Na/Ca exchange blocked the increased incidence of diastolic calcium release in mutant cells. Scn8aN1768D/+ mice exhibited bradycardia compared with controls. This difference in heart rate dissipated after administration of norepinephrine, and there were no differences in heart rate in denervated ex vivo hearts, implicating parasympathetic hyperexcitability in the Scn8aN1768D/+ animals. When challenged with norepinephrine and caffeine to simulate a catecholaminergic surge, Scn8aN1768D/+ mice showed ventricular arrhythmias. Two of three mutant mice under continuous ECG telemetry recording experienced death, with severe bradycardia preceding asystole. Thus, in addition to central neuron hyperexcitability, Scn8aN1768D/+ mice have cardiac myoycte and parasympathetic neuron hyperexcitability. Simultaneous dysfunction in these systems may contribute to SUDEP associated with mutations of Scn8a.

Cannabinoid-induced depression of synaptic transmission is switched to stimulation when dopaminergic tone is increased in the globus pallidus of the rodent.

Because activation of D2 receptors reverses the neurochemical effects of cannabinoids, we examined whether increasing dopaminergic tone in the globus pallidus (GPe) switches cannabinoid induced depression of synaptic transmission. GABAergic synaptic currents evoked in pallidal neurons by stimulation of striatal projections (IPSCs) were depressed by perfusion with the CB1R agonist ACEA. Coactivation of D2Rs with quinpirole converted the depression into stimulation. Pretreatment with pertussis toxin (PTX) to limit Gi/o protein coupling also switched the CB1R-induced depression of IPSCs. The stimulation of IPSCs was blocked by the selective PKA blocker H89. Changes in the paired pulse ratio during both inhibitory and stimulatory responses indicate that the effects are due to changes in transmitter release. Postsynaptic depolarization induces endocannabinoid release that inhibits transmitter release (DSI). When D2Rs were activated with quinpirole, depolarization increased transmission instead of depressing it. This increase was blocked by AM251. We also examined the effects of CB1R/D2R coactivation on cAMP accumulation in the GPe to further verify that the AC/PKA cascade is involved. CB1R/D2R coactivation converted the inhibition of cAMP seen when each receptor is stimulated alone into a stimulation. We also determined the effects on turning behavior of unilateral injection of ACEA into the GPe of awake animals and its modification by dopamine antagonists. Blockade of D2 family receptors with sulpiride antagonized the motor effects of ACEA. We show, for the first time, that cannabinoid-inhibition of synaptic transmission in the GPe becomes a stimulation after D2Rs or PTX treatment and that the switch is probably relevant for the control of motor behavior.

Eating 'Junk-Food' Produces Rapid and Long-Lasting Increases in NAc CP-AMPA Receptors: Implications for Enhanced Cue-Induced Motivation and Food Addiction.

Urges to eat are influenced by stimuli in the environment that are associated with food (food cues). Obese people are more sensitive to food cues, reporting stronger craving and consuming larger portions after food cue exposure. The nucleus accumbens (NAc) mediates cue-triggered motivational responses, and activations in the NAc triggered by food cues are stronger in people who are susceptible to obesity. This has led to the idea that alterations in NAc function similar to those underlying drug addiction may contribute to obesity, particularly in obesity-susceptible individuals. Motivational responses are mediated in part by NAc AMPA receptor (AMPAR) transmission, and recent work shows that cue-triggered motivation is enhanced in obesity-susceptible rats after 'junk-food' diet consumption. Therefore, here we determined whether NAc AMPAR expression and function is increased by 'junk-food' diet consumption in obesity-susceptible vs -resistant populations using both outbred and selectively bred models of susceptibility. In addition, cocaine-induced locomotor activity was used as a general 'read out' of mesolimbic function after 'junk-food' consumption. We found a sensitized locomotor response to cocaine in rats that gained weight on a 'junk-food' diet, consistent with greater responsivity of mesolimbic circuits in obesity-susceptible groups. In addition, eating 'junk-food' increased NAc calcium-permeable-AMPAR (CP-AMPAR) function only in obesity-susceptible rats. This increase occurred rapidly, persisted for weeks after 'junk-food' consumption ceased, and preceded the development of obesity. These data are considered in light of enhanced cue-triggered motivation and striatal function in obesity-susceptible rats and the role of NAc CP-AMPARs in enhanced motivation and addiction.

Dravet syndrome patient-derived neurons suggest a novel epilepsy mechanism.

OBJECTIVE: Neuronal channelopathies cause brain disorders, including epilepsy, migraine, and ataxia. Despite the development of mouse models, pathophysiological mechanisms for these disorders remain uncertain. One particularly devastating channelopathy is Dravet syndrome (DS), a severe childhood epilepsy typically caused by de novo dominant mutations in the SCN1A gene encoding the voltage-gated sodium channel Na(v) 1.1. Heterologous expression of mutant channels suggests loss of function, raising the quandary of how loss of sodium channels underlying action potentials produces hyperexcitability. Mouse model studies suggest that decreased Na(v) 1.1 function in interneurons causes disinhibition. We aim to determine how mutant SCN1A affects human neurons using the induced pluripotent stem cell (iPSC) method to generate patient-specific neurons. METHODS: Here we derive forebrain-like pyramidal- and bipolar-shaped neurons from 2 DS subjects and 3 human controls by iPSC reprogramming of fibroblasts. DS and control iPSC-derived neurons are compared using whole-cell patch clamp recordings. Sodium current density and intrinsic neuronal excitability are examined. RESULTS: Neural progenitors from DS and human control iPSCs display a forebrain identity and differentiate into bipolar- and pyramidal-shaped neurons. DS patient-derived neurons show increased sodium currents in both bipolar- and pyramidal-shaped neurons. Consistent with increased sodium currents, both types of patient-derived neurons show spontaneous bursting and other evidence of hyperexcitability. Sodium channel transcripts are not elevated, consistent with a post-translational mechanism. INTERPRETATION: These data demonstrate that epilepsy patient-specific iPSC-derived neurons are useful for modeling epileptic-like hyperactivity. Our findings reveal a previously unrecognized cell-autonomous epilepsy mechanism potentially underlying DS, and offer a platform for screening new antiepileptic therapies.

Identification of the cysteine residue responsible for disulfide linkage of Na+ channel α and ß2 subunits.

Voltage-gated Na(+) channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked ß subunit (ß1 or ß3), and one disulfide-linked ß subunit (ß2 or ß4). The final step in Na(+) channel biosynthesis in central neurons is concomitant α-ß2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding ß2) null mice have reduced Na(+) channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant ß2 cDNA constructs to investigate the cysteine residue(s) responsible for α-ß2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between α and ß2 subunits. Loss of α-ß2 covalent complex formation disrupts the targeting of ß2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with α is required for normal ß2 subcellular localization in vivo. WT ß2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant ß2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-ß2 covalent association via a single, extracellular disulfide bond is required for ß2 targeting to specialized neuronal subcellular domains and for ß2 association with the neuronal cytoskeleton within those domains.

Voltage-gated Na+ channel ß1B: a secreted cell adhesion molecule involved in human epilepsy.

Scn1b-null mice have a severe neurological and cardiac phenotype. Human mutations in SCN1B result in epilepsy and cardiac arrhythmia. SCN1B is expressed as two developmentally regulated splice variants, ß1 and ß1B, that are each expressed in brain and heart in rodents and humans. Here, we studied the structure and function of ß1B and investigated a novel human SCN1B epilepsy-related mutation (p.G257R) unique to ß1B. We show that wild-type ß1B is not a transmembrane protein, but a soluble protein expressed predominantly during embryonic development that promotes neurite outgrowth. Association of ß1B with voltage-gated Na+ channels Na(v)1.1 or Na(v)1.3 is not detectable by immunoprecipitation and ß1B does not affect Na(v)1.3 cell surface expression as measured by [(3)H]saxitoxin binding. However, ß1B coexpression results in subtle alteration of Na(v)1.3 currents in transfected cells, suggesting that ß1B may modulate Na+ current in brain. Similar to the previously characterized p.R125C mutation, p.G257R results in intracellular retention of ß1B, generating a functional null allele. In contrast, two other SCN1B mutations associated with epilepsy, p.C121W and p.R85H, are expressed at the cell surface. We propose that ß1B p.G257R may contribute to epilepsy through a mechanism that includes intracellular retention resulting in aberrant neuronal pathfinding.

Na+ channel Scn1b gene regulates dorsal root ganglion nociceptor excitability in vivo.

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na(+) current (I(Na)) mediated by voltage-gated Na(+) channels (VGSCs). In nociceptive DRG neurons, VGSC ß2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by ß1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S I(Na) inactivation, reduced persistent TTX-R I(Na), a prolonged rate of recovery of TTX-R I(Na) from inactivation, and reduced cell surface expression of Na(v)1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K(+) current (I(to)) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both I(Na) and I(K).

A functional null mutation of SCN1B in a patient with Dravet syndrome.

Dravet syndrome (also called severe myoclonic epilepsy of infancy) is one of the most severe forms of childhood epilepsy. Most patients have heterozygous mutations in SCN1A, encoding voltage-gated sodium channel Na(v)1.1 alpha subunits. Sodium channels are modulated by beta1 subunits, encoded by SCN1B, a gene also linked to epilepsy. Here we report the first patient with Dravet syndrome associated with a recessive mutation in SCN1B (p.R125C). Biochemical characterization of p.R125C in a heterologous system demonstrated little to no cell surface expression despite normal total cellular expression. This occurred regardless of coexpression of Na(v)1.1 alpha subunits. Because the patient was homozygous for the mutation, these data suggest a functional SCN1B null phenotype. To understand the consequences of the lack of beta1 cell surface expression in vivo, hippocampal slice recordings were performed in Scn1b(-/-) versus Scn1b(+/+) mice. Scn1b(-/-) CA3 neurons fired evoked action potentials with a significantly higher peak voltage and significantly greater amplitude compared with wild type. However, in contrast to the Scn1a(+/-) model of Dravet syndrome, we found no measurable differences in sodium current density in acutely dissociated CA3 hippocampal neurons. Whereas Scn1b(-/-) mice seize spontaneously, the seizure susceptibility of Scn1b(+/-) mice was similar to wild type, suggesting that, like the parents of this patient, one functional SCN1B allele is sufficient for normal control of electrical excitability. We conclude that SCN1B p.R125C is an autosomal recessive cause of Dravet syndrome through functional gene inactivation.

The voltage-gated Na+ channel beta3 subunit does not mediate trans homophilic cell adhesion or associate with the cell adhesion molecule contactin.

Voltage-gated Na(+) channel (VGSC) beta1 and beta2 subunits are multifunctional, serving as both channel modulators and cell adhesion molecules (CAMs). The purpose of this study was to determine whether VGSC beta3 subunits function as CAMs. The beta3 extracellular domain is highly homologous to beta1, suggesting that beta3 may also be a functional CAM. We investigated the trans homophilic cell adhesive properties of beta3, its association with the beta1-interacting CAM contactin, as well as its ability to interact with the cytoskeletal protein ankyrin. Our results demonstrate that, unlike beta1, beta3 does not participate in trans homophilic cell-cell adhesion or associate with contactin. Further, beta3 does not associate with ankyrin(G) in a heterologous system. Previous studies have shown that beta3 interacts with the CAM neurofascin-186 but not with VGSC beta1. Taken together, these findings suggest that, although beta1 and beta3 exhibit similar channel modulatory properties in heterologous systems, these subunits differ with regard to their homophilic and heterophilic CAM binding profiles.