Scn8a Antisense Oligonucleotide Is Protective in Mouse Models of SCN8A Encephalopathy and Dravet Syndrome.

OBJECTIVE: SCN8A encephalopathy is a developmental and epileptic encephalopathy (DEE) caused by de novo gain-of-function mutations of sodium channel Nav 1.6 that result in neuronal hyperactivity. Affected individuals exhibit early onset drug-resistant seizures, developmental delay, and cognitive impairment. This study was carried out to determine whether reducing the abundance of the Scn8a transcript with an antisense oligonucleotide (ASO) would delay seizure onset and prolong survival in a mouse model of SCN8A encephalopathy. METHODS: ASO treatment was tested in a conditional mouse model with Cre-dependent expression of the pathogenic patient SCN8A mutation p.Arg1872Trp (R1872W). This model exhibits early onset of seizures, rapid progression, and 100% penetrance. An Scn1a +/- haploinsufficient mouse model of Dravet syndrome was also treated. ASO was administered by intracerebroventricular injection at postnatal day 2, followed in some cases by stereotactic injection at postnatal day 30. RESULTS: We observed a dose-dependent increase in length of survival from 15 to 65 days in the Scn8a-R1872W/+ mice treated with ASO. Electroencephalographic recordings were normal prior to seizure onset. Weight gain and activity in an open field were unaffected, but treated mice were less active in a wheel running assay. A single treatment with Scn8a ASO extended survival of Dravet syndrome mice from 3 weeks to >5 months. INTERPRETATION: Reduction of Scn8a transcript by 25 to 50% delayed seizure onset and lethality in mouse models of SCN8A encephalopathy and Dravet syndrome. Reduction of SCN8A transcript is a promising approach to treatment of intractable childhood epilepsies. Ann Neurol 2020;87:339-346.

Pharmacologically Targeting the Fibroblast Growth Factor 14 Interaction Site on the Voltage-Gated Na+ Channel 1.6 Enables Isoform-Selective Modulation.

Voltage-gated Na+ (Nav) channels are the primary molecular determinant of the action potential. Among the nine isoforms of the Nav channel α subunit that have been described (Nav1.1-Nav1.9), Nav1.1, Nav1.2, and Nav1.6 are the primary isoforms expressed in the central nervous system (CNS). Crucially, these three CNS Nav channel isoforms display differential expression across neuronal cell types and diverge with respect to their subcellular distributions. Considering these differences in terms of their localization, the CNS Nav channel isoforms could represent promising targets for the development of targeted neuromodulators. However, current therapeutics that target Nav channels lack selectivity, which results in deleterious side effects due to modulation of off-target Nav channel isoforms. Among the structural components of the Nav channel α subunit that could be pharmacologically targeted to achieve isoform selectivity, the C-terminal domains (CTD) of Nav channels represent promising candidates on account of displaying appreciable amino acid sequence divergence that enables functionally unique protein-protein interactions (PPIs) with Nav channel auxiliary proteins. In medium spiny neurons (MSNs) of the nucleus accumbens (NAc), a critical brain region of the mesocorticolimbic circuit, the PPI between the CTD of the Nav1.6 channel and its auxiliary protein fibroblast growth factor 14 (FGF14) is central to the generation of electrical outputs, underscoring its potential value as a site for targeted neuromodulation. Focusing on this PPI, we previously developed a peptidomimetic derived from residues of FGF14 that have an interaction site on the CTD of the Nav1.6 channel. In this work, we show that whereas the compound displays dose-dependent effects on the activity of Nav1.6 channels in heterologous cells, the compound does not affect Nav1.1 or Nav1.2 channels at comparable concentrations. In addition, we show that the compound correspondingly modulates the action potential discharge and the transient Na+ of MSNs of the NAc. Overall, these results demonstrate that pharmacologically targeting the FGF14 interaction site on the CTD of the Nav1.6 channel is a strategy to achieve isoform-selective modulation, and, more broadly, that sites on the CTDs of Nav channels interacted with by auxiliary proteins could represent candidates for the development of targeted therapeutics.

Persistent and resurgent Na+ currents in vestibular calyx afferents.

Vestibular afferent neurons convey information from hair cells in the peripheral vestibular end organs to central nuclei. Primary vestibular afferent neurons can fire action potentials at high rates and afferent firing patterns vary with the position of nerve terminal endings in vestibular neuroepithelia. Terminals contact hair cells as small bouton or large calyx endings. To investigate the role of Na+ currents (INa) in firing mechanisms, we investigated biophysical properties of INa in calyx-bearing afferents. Whole cell patch-clamp recordings were made from calyx terminals in thin slices of gerbil crista at different postnatal ages: immature [postnatal day (P)5-P8, young (P13-P15), and mature (P30-P45)]. A large transient Na+ current (INaT) was completely blocked by 300 nM tetrodotoxin (TTX) in mature calyces. In addition, INaT was accompanied by much smaller persistent Na+ currents (INaP) and distinctive resurgent Na+ currents (INaR), which were also blocked by TTX. ATX-II, a toxin that slows Na+ channel inactivation, enhanced INaP in immature and mature calyces. 4,9-Anhydro-TTX (4,9-ah-TTX), which selectively blocks Nav1.6 channels, abolished the enhanced INa in mature, but not immature, calyces. Therefore, Nav1.6 channels mediate a component of INaT and INaP in mature calyces, but are minimally expressed at early postnatal days. INaR was expressed in less than one-third of calyces at P6-P8, but expression increased with development, and in mature cristae INaR was frequently found in peripheral calyces. INaR served to increase the availability of Na+ channels following brief membrane depolarizations. In current clamp, the rate and regularity of action potential firing decreased in mature peripheral calyces following 4,9-ah-TTX application. Therefore, Nav1.6 channels are upregulated during development, contribute to INaT, INaP, and INaR, and may regulate excitability by enabling higher mean discharge rates in a subpopulation of mature calyx afferents.NEW & NOTEWORTHY Action potential firing patterns differ between groups of afferent neurons innervating vestibular epithelia. We investigated the biophysical properties of Na+ currents in specialized vestibular calyx afferent terminals during postnatal development. Mature calyces express Na+ currents with transient, persistent, and resurgent components. Nav1.6 channels contribute to resurgent Na+ currents and may enhance firing in peripheral calyx afferents. Understanding Na+ channels that contribute to vestibular nerve responses has implications for developing new treatments for vestibular dysfunction.

Aberrant expression of Nav1.6 in the cochlear nucleus correlates with salicylate-induced tinnitus in rats.

Hyperactivity in cochlear nucleus (CN) is one of the major neural correlates for tinnitus induction, yet the molecular factors that participate in the neuronal hyperexcitability remain unclear. The present study showed that acute and chronic administrations of salicylate were both capable of inducing reversible tinnitus in rats. The number of GAD 65/67-immunoreactive neurons in the AVCN and DCN was decreased, while the number of VGLUT 1/2-immunoreactive neurons in the AVCN and DCN was increased when rats were experiencing tinnitus, providing evidence for excitatory-inhibitory imbalance in CN is correlated with tinnitus. Interestingly, the expression level of Nav1.6, an important subtype of voltage-gated sodium channels was significantly increased in the DCN and AVCN of rats experiencing tinnitus, the up-regulation of Nav1.6 was returned to normal level following the disappearance of tinnitus. Double-labeling experiments revealed that Nav1.6 expression was down-regulated in the GAD 65/67-positive neurons in the DCN and AVCN of rats experiencing tinnitus. Notably, the percentage of co-localization of Nav1.6 and NeuN-labeling fusiform neurons was markedly increased in the DCN during tinnitus. These findings uncover the tinnitus-associated alteration in Nav1.6, a potential key contributor that can lead to hyperexcitability in CN and contribute to salicylate-induced tinnitus.

AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells.

Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC50 value of 15 µM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Na+ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells.

Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be targeted with cannabidiol.

Mutations in brain isoforms of voltage-gated sodium channels have been identified in patients with distinct epileptic phenotypes. Clinically, these patients often do not respond well to classic anti-epileptics and many remain refractory to treatment. Exogenous as well as endogenous cannabinoids have been shown to target voltage-gated sodium channels and cannabidiol has recently received attention for its potential efficacy in the treatment of childhood epilepsies. In this study, we further investigated the ability of cannabinoids to modulate sodium currents from wild-type and epilepsy-associated mutant voltage-gated sodium channels. We first determined the biophysical consequences of epilepsy-associated missense mutations in both Nav1.1 (arginine 1648 to histidine and asparagine 1788 to lysine) and Nav1.6 (asparagine 1768 to aspartic acid and leucine 1331 to valine) by obtaining whole-cell patch clamp recordings in human embryonic kidney 293T cells with 200 µM Navß4 peptide in the pipette solution to induce resurgent sodium currents. Resurgent sodium current is an atypical near threshold current predicted to increase neuronal excitability and has been implicated in multiple disorders of excitability. We found that both mutations in Nav1.6 dramatically increased resurgent currents while mutations in Nav1.1 did not. We then examined the effects of anandamide and cannabidiol on peak transient and resurgent currents from wild-type and mutant channels. Interestingly, we found that cannabidiol can preferentially target resurgent sodium currents over peak transient currents generated by wild-type Nav1.6 as well as the aberrant resurgent and persistent current generated by Nav1.6 mutant channels. To further validate our findings, we examined the effects of cannabidiol on endogenous sodium currents from striatal neurons, and similarly we found an inhibition of resurgent and persistent current by cannabidiol. Moreover, current clamp recordings show that cannabidiol reduces overall action potential firing of striatal neurons. These findings suggest that cannabidiol could be exerting its anticonvulsant effects, at least in part, through its actions on voltage-gated sodium channels, and resurgent current may be a promising therapeutic target for the treatment of epilepsy syndromes.

Selective blocking effects of 4,9-anhydrotetrodotoxin, purified from a crude mixture of tetrodotoxin analogues, on NaV1.6 channels and its chemical aspects.

Tetrodotoxin (TTX) is a potent neurotoxin found in a number of marine creatures including the pufferfish, where it is synthesized by bacteria and accumulated through the food chain. It is a potent and selective blocker of some types of voltage-gated Na+ channel (NaV channel). 4,9-Anhydrotetrodotoxin (4,9-anhydroTTX) was purified from a crude mixture of TTX analogues (such as TTX, 4-epiTTX, 6-epiTTX, 11-oxoTTX and 11-deoxyTTX) by the use of liquid chromatography-fluorescence detection (LC-FLD) techniques. Recently, it has been reported that 4,9-anhydroTTX selectively blocks the activity of NaV1.6 channels with a blocking efficacy 40-160 times higher than that for other TTX-sensitive NaV1.x channel isoforms. However, little attention has been paid to the molecular properties of the α-subunit in NaV1.6 channels and the characteristics of binding of 4,9-anhydroTTX. From a functional point of view, it is important to determine the relative expression of NaV1.6 channels in a wide variety of tissues. The aim of this review is to discuss briefly current knowledge about the pharmacology of 4,9-anhydroTTX, and provide an analysis of the molecular structure of native NaV1.6 channels. In addition, chemical aspects of 4,9-anhydroTTX are briefly covered.

Neurosteroids allopregnanolone sulfate and pregnanolone sulfate have diverse effect on the α subunit of the neuronal voltage-gated sodium channels Nav1.2, Nav1.6, Nav1.7, and Nav1.8 expressed in xenopus oocytes.

BACKGROUND: The neurosteroids allopregnanolone and pregnanolone are potent positive modulators of γ-aminobutyric acid type A receptors. Antinociceptive effects of allopregnanolone have attracted much attention because recent reports have indicated the potential of allopregnanolone as a therapeutic agent for refractory pain. However, the analgesic mechanisms of allopregnanolone are still unclear. Voltage-gated sodium channels (Nav) are thought to play important roles in inflammatory and neuropathic pain, but there have been few investigations on the effects of allopregnanolone on sodium channels. METHODS: Using voltage-clamp techniques, the effects of allopregnanolone sulfate (APAS) and pregnanolone sulfate (PAS) on sodium current were examined in Xenopus oocytes expressing Nav1.2, Nav1.6, Nav1.7, and Nav1.8 α subunits. RESULTS: APAS suppressed sodium currents of Nav1.2, Nav1.6, and Nav1.7 at a holding potential causing half-maximal current in a concentration-dependent manner, whereas it markedly enhanced sodium current of Nav1.8 at a holding potential causing maximal current. Half-maximal inhibitory concentration values for Nav1.2, Nav1.6, and Nav1.7 were 12 ± 4 (n = 6), 41 ± 2 (n = 7), and 131 ± 15 (n = 5) µmol/l (mean ± SEM), respectively. The effects of PAS were lower than those of APAS. From gating analysis, two compounds increased inactivation of all α subunits, while they showed different actions on activation of each α subunit. Moreover, two compounds showed a use-dependent block on Nav1.2, Nav1.6, and Nav1.7. CONCLUSION: APAS and PAS have diverse effects on sodium currents in oocytes expressing four α subunits. APAS inhibited the sodium currents of Nav1.2 most strongly.

Evidence for a role of Nav1.6 in facilitating increases in neuronal hyperexcitability during epileptogenesis.

During epileptogenesis a series of molecular and cellular events occur, culminating in an increase in neuronal excitability, leading to seizure initiation. The entorhinal cortex has been implicated in the generation of epileptic seizures in both humans and animal models of temporal lobe epilepsy. This hyperexcitability is due, in part, to proexcitatory changes in ion channel activity. Sodium channels play an important role in controlling neuronal excitability, and alterations in their activity could facilitate seizure initiation. We sought to investigate whether medial entorhinal cortex (mEC) layer II neurons become hyperexcitable and display proexcitatory behavior of Na channels during epileptogenesis. Experiments were conducted 7 days after electrical induction of status epilepticus (SE), a time point during the latent period of epileptogenesis and before the onset of seizures. mEC layer II stellate neurons from post-SE animals were hyperexcitable, eliciting action potentials at higher frequencies compared with control neurons. Na channel currents recorded from post-SE neurons revealed increases in Na current amplitudes, particularly persistent and resurgent currents, as well as depolarized shifts in inactivation parameters. Immunocytochemical studies revealed increases in voltage-gated Na (Nav) 1.6 isoform levels. The toxin 4,9-anhydro-tetrodotoxin, which has greater selectivity for Nav1.6 over other Na channel isoforms, suppressed neuronal hyperexcitability, reduced macroscopic Na currents, persistent and resurgent Na current densities, and abolished depolarized shifts in inactivation parameters in post-SE neurons. These studies support a potential role for Nav1.6 in facilitating the hyperexcitability of mEC layer II neurons during epileptogenesis.

Resurgent-like currents in mouse vas deferens myocytes are mediated by NaV1.6 voltage-gated sodium channels.

Patch-clamp experiments were performed to investigate the molecular properties of resurgent-like currents in single smooth muscle cells dispersed from mouse vas deferens, utilizing both Na(V)1.6-null mice (Na(V)1.6(-/-)), lacking the expression of the Scn8a Na(+) channel gene, and their wild-type littermates (Na(V)1.6(+/+)). Na(V)1.6 immunoreactivity was clearly visible in dispersed smooth muscle cells obtained from Na(V)1.6(+/+), but not Na(V)1.6(-/-), vas deferens. Following a depolarization to +30 mV from a holding potential of -70 mV (to produce maximal inactivation of the Na(+) current), repolarization to voltages between -60 and +20 mV elicited a tetrodotoxin (TTX)-sensitive inward current in Na(V)1.6(+/+), but not Na(V)1.6(-/-), vas deferens myocytes. The resurgent-like current in Na(V)1.6(+/+) vas deferens myocytes peaked at approximately -20 mV in the current-voltage relationship. The peak amplitude of the resurgent-like current remained at a constant level when the membrane potential was repolarized to -20 mV following the application of depolarizing rectangular pulses to more positive potentials than +20 mV. 4,9-Anhydrotetrodotoxin (4,9-anhydroTTX), a selective Na(V)1.6 blocking toxin, purified from a crude mixture of TTX analogues by LC-FLD techniques, reversibly suppressed the resurgent-like currents. ß-Pompilidotoxin, a voltage-gated Na(+) channel activator, evoked sustained resurgent-like currents in Na(V)1.6(+/+) but not Na(V)1.6(-/-) murine vas deferens myocytes. These results strongly indicate that, primarily, resurgent-like currents are generated as a result of Na(V)1.6 channel activity.