Dataset Information

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-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.

SUBMITTER: Weuring WJ

PROVIDER: S-EPMC7058281 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Weuring Wout J WJ Singh Sakshi S Volkers Linda L Rook Martin B MB van 't Slot Ruben H RH Bosma Marjolein M Inserra Marco M Vetter Irina I Verhoeven-Duif Nanda M NM Braun Kees P J KPJ Rivara Mirko M Koeleman Bobby P C BPC

PloS one 20200305 3

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 eith ...[more]

PMID: 32134913

Similar Datasets

Project description:Variants implicated in childhood epilepsy have been identified in all four voltage-gated sodium channels that initiate action potentials in the central nervous system. Previous research has focused on the functional effects of particular variants within the most studied of these channels (NaV1.1, NaV1.2 and NaV1.6); however, there have been few comparative studies across channels to infer the impact of mutations in patients with epilepsy. Here we compare patterns of variation in patient and public databases to test the hypothesis that regions of known functional significance within voltage-gated sodium (NaV) channels have an increased burden of deleterious variants. We assessed mutational burden in different regions of the Nav channels by (1) performing Fisher exact tests on odds ratios to infer excess variants in domains, segments, and loops of each channel in patient databases versus public "control" databases, and (2) comparing the cumulative distribution of variant sites along DNA sequences of each gene in patient and public databases (i.e., independent of protein structure). Patient variant density was concordant among channels in regions known to play a role in channel function, with statistically significant higher patient variant density in S4-S6 and DIII-DIV and an excess of public variants in SI-S3, DI-DII, DII-DIII. On the other hand, channel-specific patterns of patient burden were found in the NaV1.6 inactivation gate and NaV1.1 S5-S6 linkers, while NaV1.2 and NaV1.6 S4-S5 linkers and S5 segments shared patient variant patterns that contrasted with those in NaV1.1. These different patterns may reflect different roles played by the NaV1.6 inactivation gate in action potential propagation, and by NaV1.1 S5-S6 linkers in loss of function and haploinsufficiency. Interestingly, NaV1.2 and NaV1.6 both lack amino acid substitutions over significantly long stretches in both the patient and public databases suggesting that new mutations in these regions may cause embryonic lethality or a non-epileptic disease phenotype.

Project description:Dravet syndrome is a developmental and epileptic encephalopathy characterized by seizures, behavioral abnormalities, developmental deficits, and elevated risk of sudden unexpected death in epilepsy (SUDEP). Most patient cases are caused by de novo loss-of-function mutations in the gene SCN1A, causing a haploinsufficiency of the alpha subunit of the voltage-gated sodium channel NaV1.1. Within the brain, NaV1.1 is primarily localized to the axons of inhibitory neurons, and decreased NaV1.1 function is hypothesized to reduce GABAergic inhibitory neurotransmission within the brain, driving neuronal network hyperexcitability and subsequent pathology. We have developed a human in vitro model of Dravet syndrome using differentiated neurons derived from patient iPSC and enriched for GABA expressing neurons. Neurons were plated on high definition multielectrode arrays (HD-MEAs), permitting recordings from the same cultures over the 7-weeks duration of study at the network, single cell, and subcellular resolution. Using this capability, we characterized the features of axonal morphology and physiology. Neurons developed increased spiking activity and synchronous network bursting. Recordings were processed through a spike sorting pipeline for curation of single unit activity and to assess the effects of pharmacological treatments. At 7-weeks, the application of the GABAAR receptor agonist muscimol eliminated network bursting, indicating the presence of GABAergic neurotransmission. To identify the role of NaV1.1 on neuronal and network activity, cultures were treated with a dose-response of the NaV1.1 potentiator δ-theraphotoxin-Hm1a. This resulted in a strong increase in firing rates of putative GABAergic neurons, an increase in the intraburst firing rate, and eliminated network bursting. These results validate that potentiation of NaV1.1 in Dravet patient iPSC-derived neurons results in decreased firing synchrony in neuronal networks through increased GABAergic neuron activity and support the use of human neurons and HD-MEAs as viable high-throughput electrophysiological platform to enable therapeutic discovery.

Project description:Current antiepileptic drugs (AEDs) are undesirable for many reasons including the inability to reduce seizures in certain types of epilepsy, such as Dravet syndrome (DS) where in one-third of patients does not respond to current AEDs, and severe adverse effects that are frequently experienced by patients. Epidiolex, a cannabidiol (CBD)-based drug, was recently approved for treatment of DS. While Epidiolex shows great promise in reducing seizures in patients with DS, it is used in conjunction with other AEDs and can cause liver toxicity. To investigate whether other cannabis-derived compounds could also reduce seizures, the antiepileptic effects of CBD, Δ9-tetrahydrocannabinol (THC), cannabidivarin (CBDV), cannabinol (CBN), and linalool (LN) were compared in both a chemically-induced (pentylenetetrazole, PTZ) and a DS (scn1Lab-/-) seizure models. Zebrafish (Danio rerio) that were either wild-type (Tupfel longfin) or scn1Lab-/- (DS) were exposed to CBD, THC, CBDV, CBN, or LN for 24 h from 5 to 6 days postfertilization. Following exposure, total distance traveled was measured in a ViewPoint Zebrabox to determine if these compounds reduced seizure-like activity. Cannabidiol (0.6 and 1 μM) and THC (1 and 4 μM) significantly reduced PTZ-induced total distance moved. At the highest THC concentration, the significant reduction in PTZ-induced behavior was likely the result of sedation as opposed to antiseizure activity. In the DS model, CBD (0.6 μM), THC (1 μM), CBN (0.6 and 1 μM), and LN (4 μM) significantly reduced total distance traveled. Cannabinol was the most effective at reducing total distance relative to controls. In addition to CBD, other cannabis-derived compounds showed promise in reducing seizure-like activity in zebrafish. Specifically, four of the five compounds were effective in the DS model, whereas in the PTZ model, only CBD and THC were, suggesting a divergence in the mode of action among the cannabis constituents.

Project description:Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs). Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navβ4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navβ4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navβ4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navβ4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navβ4 peptide. Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have distinct biophysical properties that may differentially contribute to regulating neuronal excitability.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets