Prominent role of forebrain excitatory neurons in SCN8A encephalopathy.

De novo mutations of the sodium channel gene SCN8A result in an epileptic encephalopathy with refractory seizures, developmental delay, and elevated risk of sudden death. p.Arg1872Trp is a recurrent de novo SCN8A mutation reported in 14 unrelated individuals with epileptic encephalopathy that included seizure onset in the prenatal or infantile period and severe verbal and ambulatory comorbidities. The major biophysical effect of the mutation was previously shown to be impaired channel inactivation accompanied by increased current density. We have generated a conditional mouse mutation in which expression of this severe gain-of-function mutation is dependent upon Cre recombinase. Global activation of p.Arg1872Trp by EIIa-Cre resulted in convulsive seizures and lethality at 2 weeks of age. Neural activation of the p.Arg1872Trp mutation by Nestin-Cre also resulted in early onset seizures and death. Restriction of p.Arg1872Trp expression to excitatory neurons using Emx1-Cre recapitulated seizures and juvenile lethality between 1 and 2 months of age. In contrast, activation of p.Arg1872Trp in inhibitory neurons by Gad2-Cre or Dlx5/6-Cre did not induce seizures or overt neurological dysfunction. The sodium channel modulator GS967/Prax330 prolonged survival of mice with global expression of R1872W and also modulated the activity of the mutant channel in transfected cells. Activation of the p.Arg1872Trp mutation in adult mice was sufficient to generate seizures and death, indicating that successful therapy will require lifelong treatment. These findings provide insight into the pathogenic mechanism of this gain-of-function mutation of SCN8A and identify excitatory neurons as critical targets for therapeutic intervention.

CACHD1 is an α2δ-Like Protein That Modulates CaV3 Voltage-Gated Calcium Channel Activity.

The putative cache (Ca2+ channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the α2δ voltage-gated Ca2+ channel auxiliary subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus, and cerebellum, with a broadly similar tissue distribution to CaV3 subunits, in particular CaV3.1. In expression studies, CACHD1 increased cell-surface localization of CaV3.1, and these proteins were in close proximity at the cell surface, consistent with the formation of CACHD1-CaV3.1 complexes. In functional electrophysiological studies, coexpression of human CACHD1 with CaV3.1, CaV3.2, and CaV3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, α2δ-1 had no effect on peak current density or maximal conductance in CaV3.1, CaV3.2, or CaV3.3. A comparison of CACHD1-mediated increases in CaV3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female embryonic day 19 rats, CACHD1 overexpression increased CaV3-mediated action potential firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an α2δ-like protein that functionally modulates CaV3 voltage-gated calcium channel activity.SIGNIFICANCE STATEMENT This is the first study to characterize the Ca2+ channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is widely expressed in the CNS, in particular in the thalamus, hippocampus, and cerebellum. CACHD1 distribution is similar to that of low voltage-activated (CaV3, T-type) calcium channels, in particular to CaV3.1, a protein that regulates neuronal excitability and is a potential therapeutic target in conditions such as epilepsy and pain. CACHD1 is structurally an α2δ-like protein that functionally increases CaV3 calcium current. CACHD1 increases the presence of CaV3.1 at the cell surface, forms complexes with CaV3.1 at the cell surface, and causes an increase in channel open probability. In hippocampal neurons, CACHD1 causes increases in neuronal firing. Thus, CACHD1 represents a novel protein that modulates CaV3 activity.

Aberrant Sodium Channel Currents and Hyperexcitability of Medial Entorhinal Cortex Neurons in a Mouse Model of SCN8A Encephalopathy.

SCN8A encephalopathy, or early infantile epileptic encephalopathy 13 (EIEE13), is caused predominantly by de novo gain-of-function mutations in the voltage-gated Na channel Nav1.6. Affected individuals suffer from refractory seizures, developmental delay, cognitive disability, and elevated risk of sudden unexpected death in epilepsy (SUDEP). A knock-in mouse model carrying the patient mutation p.Asn1768Asp (N1768D) reproduces many features of the disorder, including spontaneous seizures and SUDEP. We used the mouse model to examine the effects of the mutation on layer II stellate neurons of the medial entorhinal cortex (mEC), which transmit excitatory input to the hippocampus. Heterozygous (Scn8aD/+), homozygous (Scn8aD/D)), and WT (Scn8a+/+) littermates were compared at 3 weeks of age, the time of seizure onset for homozygous mice. Heterozygotes remain seizure free for another month. mEC layer II neurons of heterozygous and homozygous mice were hyperexcitable and generated long-lasting depolarizing potentials with bursts of action potentials after synaptic stimulation. Recording of Na currents revealed proexcitatory increases in persistent and resurgent currents and rightward shifts in inactivation parameters, leading to significant increases in the magnitude of window currents. The proexcitatory changes were more pronounced in homozygous mice than in heterozygotes, consistent with the earlier age of seizure onset in homozygotes. These studies demonstrate that the N1768D mutation increases the excitability of mEC layer II neurons by increasing persistent and resurgent Na currents and disrupting channel inactivation. The aberrant activities of mEC layer II neurons would provide excessive excitatory input to the hippocampus and contribute to hyperexcitability of hippocampal neurons in this model of SCN8A encephalopathy.SIGNIFICANCE STATEMENTSCN8A encephalopathy is a devastating neurological disorder that results from de novo mutations in the Na channel Nav1.6. In addition to seizures, patients suffer from cognitive and developmental delays and are at high risk for sudden unexpected death in epilepsy (SUDEP). A mouse knock-in model expressing the patient mutation N1768D reproduces several pathological phenotypes, including spontaneous seizures and sudden death. We demonstrate that medial entorhinal cortex (mEC) neurons from the mouse model exhibit proexcitatory alterations in Na channel activity, some of which were not seen in hippocampal or cortical neurons, and resulting in neuronal hyperexcitability. Because mEC neurons regulate the activity of the hippocampus, which plays an important role in seizure onset, we propose that these profound changes in mEC neuron excitability associated with the gain-of-function mutation of Nav1.6 may increase excitatory drive into the hippocampus, culminating in seizure activity and SUDEP.

Partial loss-of-function of sodium channel SCN8A in familial isolated myoclonus.

Variants in the neuronal sodium channel gene SCN8A have been implicated in several neurological disorders. Early infantile epileptic encephalopathy type 13 results from de novo gain-of-function mutations that alter the biophysical properties of the channel. Complete loss-of-function variants of SCN8A have been identified in cases of isolated intellectual disability. We now report a novel heterozygous SCN8A variant, p.Pro1719Arg, in a small pedigree with five family members affected with autosomal dominant upper limb isolated myoclonus without seizures or cognitive impairment. Functional analysis of the p.Pro1719Arg variant in transfected neuron-derived cells demonstrated greatly reduced Nav 1.6 channel activity without altered gating properties. Hypomorphic alleles of Scn8a in the mouse are known to result in similar movement disorders. This study expands the phenotypic and functional spectrum of SCN8A variants to include inherited nonepileptic isolated myoclonus. SCN8A can be considered as a candidate gene for isolated movement disorders without seizures.

Pro-excitatory alterations in sodium channel activity facilitate subiculum neuron hyperexcitability in temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is a common form of adult epilepsy involving the limbic structures of the temporal lobe. Subiculum neurons act to provide a major output from the hippocampus and consist of a large population of endogenously bursting excitatory neurons. In TLE, subiculum neurons are largely spared, become hyperexcitable and show spontaneous epileptiform activity. The basis for this hyperexcitability is unclear, but is likely to involve alterations in the expression levels and function of various ion channels. In this study, we sought to determine the importance of sodium channel currents in facilitating neuronal hyperexcitability of subiculum neurons in the continuous hippocampal stimulation (CHS) rat model of TLE. Subiculum neurons from TLE rats were hyperexcitable, firing a higher frequency of action potentials after somatic current injection and action potential (AP) bursts after synaptic stimulation. Voltage clamp recordings revealed increases in resurgent (INaR) and persistent (INaP) sodium channel currents and pro-excitatory shifts in sodium channel activation and inactivation parameters that would facilitate increases in AP generation. Attenuation of INaR and INaP currents with 4,9-anhydro-tetrodotoxin (4,9-ah TTX; 100nM), a toxin with increased potency against Nav1.6 channels, suppressed neuronal firing frequency and inhibited AP bursting induced by synaptic stimulation in TLE neurons. These findings support an important role of sodium channels, particularly Nav1.6, in facilitating subiculum neuron hyperexcitability in TLE and provide further support for the importance of INaR and INaP currents in establishing epileptiform activity of subiculum neurons.

The SCN8A encephalopathy mutation p.Ile1327Val displays elevated sensitivity to the anticonvulsant phenytoin.

OBJECTIVE: SCN8A encephalopathy (early infantile epileptic encephalopathy; EIEE13) is caused by gain-of-function mutations resulting in hyperactivity of the voltage-gated sodium channel Nav 1.6. The channel is concentrated at the axon initial segment (AIS) and is involved in establishing neuronal excitability. Clinical features of SCN8A encephalopathy include seizure onset between 0 and 18 months of age, intellectual disability, and developmental delay. Seizures are often refractory to treatment with standard antiepileptic drugs, and sudden unexpected death in epilepsy (SUDEP) has been reported in approximately 10% of patients. In a recent study, high doses of phenytoin were effective in four patients with SCN8A encephalopathy. In view of this observation, we have investigated the relationship between the functional effect of the SCN8A mutation p.Ile1327Val and its response to phenytoin. METHODS: The mutation was introduced into the Scn8a cDNA by site-directed mutagenesis. Channel activity was characterized in transfected ND7/23 cells. The effects of phenytoin (100 µm) on mutant and wild-type (WT) channels were compared. RESULTS: Channel activation parameters were shifted in a hyperpolarizing direction in the mutant channel, whereas inactivation parameters were shifted in a depolarizing direction, increasing Na channel window current. Macroscopic current decay was slowed in I1327V channels, indicating an impairment in the transition from open state to inactivated state. Channel deactivation was also delayed, allowing more channels to remain in the open state. Phenytoin (100 µm) resulted in hyperpolarized activation and inactivation curves as well as greater tonic block and use-dependent block of I1327V mutant channels relative to WT. SIGNIFICANCE: SCN8A - I1327V is a gain-of-function mutation with altered features that are predicted to increase neuronal excitability and seizure susceptibility. Phenytoin is an effective inhibitor of the mutant channel and may be of use in treating patients with gain-of-function mutations of SCN8A.

The Kv7 Modulator, Retigabine, is an Efficacious Antiseizure Drug for Delayed Treatment of Organophosphate-induced Status Epilepticus.

Benzodiazepines are the primary treatment option for organophosphate (OP)-induced status epilepticus (SE), but these antiseizure drugs (ASDs) lose efficacy as treatment is delayed. In the event of a mass civilian or military exposure, significant treatment delays are likely. New ASDs that combat benzodiazepine-resistant, OP-induced SE are critically needed, particularly if they can be efficacious after a long treatment delay. This study evaluated the efficacy of the Kv7 channel modulator, retigabine, as a novel therapy for OP-induced SE. Adult, male rats were exposed to soman or diisopropyl fluorophosphate (DFP) to elicit SE and monitored by electroencephalogram (EEG) recording. Retigabine was administered alone or adjunctive to midazolam (MDZ) at delays of 20- or 40-min in the soman model, and 60-min in the DFP model. Following EEG recordings, rats were euthanized and brain tissue was collected for Fluoro-Jade B (FJB) staining to quantify neuronal death. In the DFP model, MDZ + 15 mg/kg retigabine suppressed seizure activity and was neuroprotective. In the soman model, MDZ + 30 mg/kg retigabine suppressed seizures at 20- and 40-min delays. Without MDZ, 15 mg/kg retigabine provided partial antiseizure and neuroprotectant efficacy in the DFP model, while 30 mg/kg without MDZ failed to attenuate soman-induced SE. At 60 mg/kg, retigabine without MDZ strongly reduced seizure activity and neuronal degeneration against soman-induce SE. This study demonstrates the antiseizure and neuroprotective efficacy of retigabine against OP-induced SE. Our data suggest retigabine could be a useful adjunct to standard-of-care and has potential for use in the absence of MDZ.

Screening for Efficacious Anticonvulsants and Neuroprotectants in Delayed Treatment Models of Organophosphate-induced Status Epilepticus.

Organophosphorus (OP) compounds are deadly chemicals that exert their intoxicating effects through the irreversible inhibition of acetylcholinesterase (AChE). In addition to an excess of peripheral ailments, OP intoxication induces status epilepticus (SE) which if left untreated may lead to permanent brain damage or death. Benzodiazepines are typically the primary therapies for OP-induced SE, but these drugs lose efficacy as treatment time is delayed. The CounterACT Neurotherapeutic Screening (CNS) Program was therefore established by the National Institutes of Health (NIH) to discover novel treatments that may be administered adjunctively with the currently approved medical countermeasures for OP-induced SE in a delayed treatment scenario. The CNS program utilizes in vivo EEG recordings and Fluoro-JadeB (FJB) histopathology in two established rat models of OP-induced SE, soman (GD) and diisopropylfluorophosphate (DFP), to evaluate the anticonvulsant and neuroprotectant efficacy of novel adjunct therapies when administered at 20 or 60 min after the induction of OP-induced SE. Here we report the results of multiple compounds that have previously shown anticonvulsant or neuroprotectant efficacy in other models of epilepsy or trauma. Drugs tested were ganaxolone, diazoxide, bumetanide, propylparaben, citicoline, MDL-28170, and chloroquine. EEG analysis revealed that ganaxolone demonstrated the most robust anticonvulsant activity, whereas all other drugs failed to attenuate ictal activity in both models of OP-induced SE. FJB staining demonstrated that none of the tested drugs had widespread neuroprotective abilities. Overall these data suggest that neurosteroids may represent the most promising anticonvulsant option for OP-induced SE out of the seven unique mechanisms tested here. Additionally, these results suggest that drugs that provide significant neuroprotection from OP-induced SE without some degree of anticonvulsant activity are elusive, which further highlights the necessity to continue screening novel adjunct treatments through the CNS program.

Prax330 reduces persistent and resurgent sodium channel currents and neuronal hyperexcitability of subiculum neurons in a mouse model of SCN8A epileptic encephalopathy.

SCN8A epileptic encephalopathy is a severe genetic epilepsy syndrome caused by de novo gain-of-function mutations of SCN8A encoding the voltage-gated sodium (Na) channel (VGSC) NaV1.6. Therapeutic management is difficult in many patients, leading to uncontrolled seizures and risk of sudden unexpected death in epilepsy (SUDEP). There is a need to develop novel anticonvulsants that can specifically target aberrant VGSC activity associated with SCN8A gain-of-function mutations. In this study, we investigate the effects of Prax330, a novel VGSC inhibitor, on the biophysical properties of wild-type (WT) NaV1.6 and the patient mutation p.Asn1768Asp (N1768D) in ND7/23 cells. The effects of Prax330 on persistent (INaP) and resurgent (INaR) Na currents and neuronal excitability in subiculum neurons from a knock-in mouse model of the Scn8a-N1768D mutation (Scn8aD/+) were also examined. In ND7/23 cells, Prax330 reduced INaP currents recorded from cells expressing Scn8a-N1768D and hyperpolarized steady-state inactivation curves. Recordings from brain slices demonstrated elevated INaP and INaR in subiculum neurons from Scn8aD/+ mutant mice and abnormally large action potential (AP) burst-firing events in a subset of neurons. Prax330 (1 µM) reduced both INaP and INaR and suppressed AP bursts, with a smaller effect on AP waveforms that had similar morphology to WT neurons. Prax330 (1 µM) also reduced synaptically-evoked APs in Scn8aD/+ subiculum neurons but not in WT neurons. Our results highlight the efficacy of targeting INaP and INaR and inactivation parameters in controlling subiculum excitability and suggest Prax330 as a promising novel therapy for SCN8A epileptic encephalopathy.

Inhibition of T-Type calcium channels in mEC layer II stellate neurons reduces neuronal hyperexcitability associated with epilepsy.

Temporal lobe epilepsy (TLE) is a form of adult epilepsy involving the entorhinal cortex (EC). Layer II neurons of the medial EC (mEC) are spared and become hyperexcitable in TLE. Studies have suggested a role for T-type calcium channels (T-type Ca2+ channels) in facilitating increases in neuronal activity associated with TLE within the hippocampus. We sought to determine if T-type Ca2+ channels play a role in facilitating neuronal hyperexcitability of layer II mEC stellate neurons in TLE. TLE was induced in rats by electrical stimulation of the hippocampus to induce status epilepticus (SE). Brain slices were prepared from rats exhibiting spontaneous seizures and compared with age-matched control rats. Action potentials (APs) were evoked either by current injection steps or via presynaptic stimulation of mEC deep layers. The selective T-type Ca2+ channel antagonist, TTA-P2 (1 µM), was applied to determine the role of T-type Ca2+ channels in maintaining neuronal excitability. Quantitative PCR techniques were used to assess T-type Ca2+ channel isoform mRNA levels within the mEC layer II. TLE mEC layer II stellate neurons were hyperexcitable compared to control neurons, evoking a higher frequency of APs and generating bursts of APs when synaptically stimulated. TTA-P2 (1 µM) reduced firing frequencies in TLE and control neurons and reduced AP burst firing in TLE stellate neurons. TTA-P2 had little effect on synaptically evoked AP's in control neurons. TTA-P2 also inhibited rebound APs evoked in TLE neurons to a greater degree than in control neurons. TLE tissue had almost a 3-fold increase in Cav3.1 mRNA compared to controls. Cav3.2 or Cav3.3 levels were unchanged. These findings support a role for T-type Ca2+ channel in establishing neuronal hyperexcitability of mEC layer II stellate neurons in TLE. Increased expression of Cav3.1 may be important for establishing neuronal hyperexcitability of mEC layer II neurons in TLE.

Loss-of-function variants of SCN8A in intellectual disability without seizures.

OBJECTIVE: To determine the functional effect of SCN8A missense mutations in 2 children with intellectual disability and developmental delay but no seizures. METHODS: Genomic DNA was analyzed by next-generation sequencing. SCN8A variants were introduced into the Nav1.6 complementary DNA by site-directed mutagenesis. Channel activity was measured electrophysiologically in transfected ND7/23 cells. The stability of the mutant channels was assessed by Western blot. RESULTS: Both children were heterozygous for novel missense variants that altered conserved residues in transmembrane segments of Nav1.6, p.Gly964Arg in D2S6 and p.Glu1218Lys in D3S1. Both altered amino acids are evolutionarily conserved in vertebrate and invertebrate channels and are predicted to be deleterious. Neither was observed in the general population. Both variants completely prevented the generation of sodium currents in transfected cells. The abundance of Nav1.6 protein was reduced by the Glu1218Lys substitution. CONCLUSIONS: Haploinsufficiency of SCN8A is associated with cognitive impairment. These observations extend the phenotypic spectrum of SCN8A mutations beyond their established role in epileptic encephalopathy (OMIM#614558) and other seizure disorders. SCN8A should be considered as a candidate gene for intellectual disability, regardless of seizure status.

Pathogenic mechanism of recurrent mutations of SCN8A in epileptic encephalopathy.

OBJECTIVE: The early infantile epileptic encephalopathy type 13 (EIEE13, OMIM #614558) results from de novo missense mutations of SCN8A encoding the voltage-gated sodium channel Nav1.6. More than 20% of patients have recurrent mutations in residues Arg1617 or Arg1872. Our goal was to determine the functional effects of these mutations on channel properties. METHODS: Clinical exome sequencing was carried out on patients with early-onset seizures, developmental delay, and cognitive impairment. Two mutations identified here, p.Arg1872Leu and p.Arg1872Gln, and two previously identified mutations, p.Arg1872Trp and p.Arg1617Gln, were introduced into Nav1.6 cDNA, and effects on electrophysiological properties were characterized in transfected ND7/23 cells. Interactions with FGF14, G-protein subunit Gßγ, and sodium channel subunit ß1 were assessed by coimmunoprecipitation. RESULTS: We identified two patients with the novel mutation p.Arg1872Leu and one patient with the recurrent mutation p.Arg1872Gln. The three mutations of Arg1872 and the mutation of Arg1617 all impaired the sodium channel transition from open state to inactivated state, resulting in channel hyperactivity. Other observed abnormalities contributing to elevated channel activity were increased persistent current, increased peak current density, hyperpolarizing shift in voltage dependence of activation, and depolarizing shift in steady-state inactivation. Protein interactions were not affected. INTERPRETATION: Recurrent mutations at Arg1617 and Arg1872 lead to elevated Nav1.6 channel activity by impairing channel inactivation. Channel hyperactivity is the major pathogenic mechanism for gain-of-function mutations of SCN8A. EIEE13 differs mechanistically from Dravet syndrome, which is caused by loss-of-function mutations of SCN1A. This distinction has important consequences for selection of antiepileptic drugs and the development of gene- and mutation-specific treatments.

Genetically targeted magnetic control of the nervous system.

Optogenetic and chemogenetic actuators are critical for deconstructing the neural correlates of behavior. However, these tools have several limitations, including invasive modes of stimulation or slow on/off kinetics. We have overcome these disadvantages by synthesizing a single-component, magnetically sensitive actuator, "Magneto," comprising the cation channel TRPV4 fused to the paramagnetic protein ferritin. We validated noninvasive magnetic control over neuronal activity by demonstrating remote stimulation of cells using in vitro calcium imaging assays, electrophysiological recordings in brain slices, in vivo electrophysiological recordings in the brains of freely moving mice, and behavioral outputs in zebrafish and mice. As proof of concept, we used Magneto to delineate a causal role of striatal dopamine receptor 1 neurons in mediating reward behavior in mice. Together our results present Magneto as an actuator capable of remotely controlling circuits associated with complex animal behaviors.