NaV1.1 and NaV1.6 selective compounds reduce the behavior phenotype and epileptiform activity in a novel zebrafish model for Dravet Syndrome

Abstract

Dravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-offunction mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating Na V 1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human Na V 1.1 or Na V 1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous zebrafish models generated by random mutagenesis or morpholino oligomers. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the GABA antagonist pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the Na V 1.1 activator AA43279 and two novel Na V 1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of spontaneous burst movements and seizure activity. Our results show that selective inhibition of Na V 1.6 could be just as efficient as selective activation of Na V 1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish

Epilepsy-Related Voltage-Gated Sodium Channelopathies: A Review

“…Phenytoin demonstrated effectiveness in decreasing seizure episodes in several patients with SCN8A-related epilepsies, however, side effects during prolonged use are very common (Boerma et al, 2016;Braakman et al, 2017). A recent study of a DS model using zebrafish demonstrated the use of the channel blocking compound MV1312, which is 5-6 fold selectivity of NaV1.6 over NaV1.1-1.7, reduced burst movement phenotype and the number of epileptiform events, activity similar to that described with the use of a selective NaV1.1 activator AA43279 (Weuring et al, 2020). Selective Nav1.6 blockers may represent a new therapeutic strategy for DS patients.…”

Distinctive Properties and Powerful Neuromodulation of Nav1.6 Sodium Channels Regulates Neuronal Excitability

“…For instance, cannabidiol and GS967 (otherwise known as Prax330) have been shown to preferentially reduce aberrant persistent and resurgent currents over transient sodium currents, however these compounds do not appear to be selective for Na v 1.6 and can target currents in other isoforms, like Na v 1.2 [ 91 , 114 , 115 , 116 ]. More recently, anti-epileptic compound screens in zebrafish models of epilepsy revealed two novel blocking compounds, MV1312 and MV1369 [ 117 ]. Although MV1312 showed a 5–6 fold selectivity of Na v 1.6 over Na v 1.1–Na v 1.7, this compound displays a comparable blocking affinity for Na v 1.8, a major PNS isoform involved in pain sensation.…”

New Challenges Resulting From the Loss of Function of Nav1.4 in Neuromuscular Diseases

“…For example, AA43279 is one small chemically synthetized compound identified in a screening campaign looking for Na v 1.1 activators. AA43279 acts as an inactivation blocker and appears efficient to functionally counteract Na v 1.1 haploinsufficiency in a zebrafish model of Dravet syndrome ( Frederiksen et al, 2017 ; Weuring et al, 2020 ). Due to their serious physiological effects and co-evolution of preys and predators, several Na v modulators including isoform-specific activators, are found in a variety of animal samples.…”

“…By screening a Food and Drug Administration (FDA)-approved compounds library, clemizole has been identified as efficient to improve the epileptic phenotype resulting from Na v 1.1 haploinsufficiency on a zebrafish model for Dravet syndrome ( Baraban et al, 2013 ). Based on the fact that Na v 1.6 GoF results in severe epileptic phenotypes, two Na v 1.6 inhibitors have also been shown to improve the phenotype in another zebrafish model of Dravet syndrome with Na v 1.1 haploinsufficiency ( Weuring et al, 2020 ).…”