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. 2020 Mar 5;15(3):e0219106.
doi: 10.1371/journal.pone.0219106. eCollection 2020.

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

Wout J Weuring  1 , Sakshi Singh  1 , Linda Volkers  2 , Martin B Rook  3 , Ruben H van 't Slot  1 , Marjolein Bosma  1 , Marco Inserra  4 , Irina Vetter  4 , Nanda M Verhoeven-Duif  1 , Kees P J Braun  5 , Mirko Rivara  6 , Bobby P C Koeleman  1
Affiliations

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

Wout J Weuring et al. PLoS One. .

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-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 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 NaV1.1 or NaV1.6 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 NaV1.1 activator AA43279 and two novel NaV1.6 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 NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems for efficacy in mammals and to screen for potential side effects.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Morphology of the Scn1Lab knockout.
A) 5 dpf Scn1Lab homozygous knockout larvae show hyperpigmentation and the absence of an inflated swim-bladder. These morphological defects are absent in heterozygous (B) or wildtype (C) zebrafish larvae.
Fig 2
Fig 2. A) Spontaneous burst movements quantified in two knockout lines using the locomotor burst movement assay B) Burst movements are unique to Scn1lab knockouts, and not caused by the absence of an inflated swimming bladder C) The burst movement phenotype of Scn1lab knockouts is partially rescued when human SCN1A, either mRNA or cDNA is introduced D) Scn1Lab knockouts show a reduction in free GABA levels E) Both heterozygote and homozygous Scn1lab knockouts show sensitivity to exposure of 5mM pentylenetetrazole F) Representative non-invasive local field potential recordings from the brain of wildtype and Scn1Lab knockout zebrafish (n = 3).
Only two types of signals can be detected in wildtype embryos: low amplitude waves and very occasional sharp single spikes from an otherwise straight and silent baseline (1 and 2). In Scn1Lab knockouts, at least three types of signal differ from wildtype recordings including trains of biphasic spikes lasting seconds (3) and several short spike events (4 and 5) which resembles epileptiform activity. Error bars = S.D. (-/-) = Scn1Lab knockout, locomotor assay n = 12 per group * <0.05 **<0.005 ***<0.0005.
Fig 3
Fig 3. Pharmacologic validation of Scn1Lab knockouts by 60 minutes (light blue) or 18 hours (dark blue) exposure of anti-epileptic drugs A) 50μM Carbamazepine B) 100μM Phenytoin C) 100μM Sodium valproate D) 12.5μM Stiripentol E) 50μM Fenfluramine F) 50μM Clemizole.
Dashed lines indicate a novel experimental plate with a seperate experimental group. Error bars = S.D., (-/-) = Scn1Lab knockout, n = 12 per group * <0.05 **<0.005 ***<0.0005.
Fig 4
Fig 4
Characterization of novel compounds and their effect on Scn1Lab knockout burst movements by 60 minute (light green) or 18 hours (dark green) exposure A) 10μM Veratridine B) 5uM AA43279 C) MV1312 show blocking selectivity for NaV1.6 over the other human ion channel subtypes but not NaV1.8. D) 5μM MV1312 E) MV1369 shows blocking selectivity for NaV1.6 over NaV1.2, NaV1.5 and NaV1.7. F) 50μM MV1369. Dashed lines indicate a novel experimental plate with a seperate experimental group. Error bars = S.D., (-/-) = Scn1Lab knockout, locomotor assays n = 12 per group, selectivity assays n = 3 per group, * <0.05 **<0.005 ***<0.0005.
Fig 5
Fig 5. Epileptiform event scoring of wildtype or Scn1Lab knockout embryos exposed to FA, AA43279 or MV1312 for 60 minutes.
Error bars = S.D n = 3 per group *
Scheme 1
Scheme 1. Reagents and conditions: 1 equiv of 2 and 5 with 4 equiv CH3COONH4 in 3.8 mL CH3OH; 1 equiv of 1 and 4 (respectively) in 3.5 mL CH3OH.
Overnight rt.
See this image and copyright information in PMC

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