Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment

Abstract

Dravet syndrome is a catastrophic pediatric epilepsy with severe intellectual disability, impaired social development and persistent drug-resistant seizures. One of its primary monogenic causes are mutations in Na_v 1.1 (SCN1A), a voltage-gated sodium channel. Here we characterize zebrafish Na_v 1.1 (scn1Lab) mutants originally identified in a chemical mutagenesis screen. Mutants exhibit spontaneous abnormal electrographic activity, hyperactivity and convulsive behaviours. Although scn1Lab expression is reduced, microarray analysis is remarkable for the small fraction of differentially expressed genes (~3%) and lack of compensatory expression changes in other scn subunits. Ketogenic diet, diazepam, valproate, potassium bromide and stiripentol attenuate mutant seizure activity; seven other antiepileptic drugs have no effect. A phenotype-based screen of 320 compounds identifies a US Food and Drug Administration-approved compound (clemizole) that inhibits convulsive behaviours and electrographic seizures. This approach represents a new direction in modelling pediatric epilepsy and could be used to identify novel therapeutics for any monogenic epilepsy disorder.

ARTICLE

Received 30 Apr 2013 | Accepted 6 Aug 2013 | Published 3 Sep 2013

Dravet syndrome is a catastrophic pediatric epilepsy with severe intellectual disability, impaired social development and persistent drug-resistant seizures. One of its primary monogenic causes are mutations in Nav1.1 (SCN1A), a voltage-gated sodium channel. Here we characterize zebrash Nav1.1 (scn1Lab) mutants originally identied in a chemical mutagenesis screen. Mutants exhibit spontaneous abnormal electrographic activity, hyperactivity and convulsive behaviours. Although scn1Lab expression is reduced, microarray analysis is remarkable for the small fraction of differentially expressed genes (B3%) and lack of compensatory expression changes in other scn subunits. Ketogenic diet, diazepam, valproate, potassium bromide and stiripentol attenuate mutant seizure activity; seven other antiepileptic drugs have no effect. A phenotype-based screen of 320 compounds identies a US Food and Drug Administration-approved compound (clemizole) that inhibits convulsive behaviours and electrographic seizures. This approach represents a new direction in modelling pediatric epilepsy and could be used to identify novel therapeutics for any monogenic epilepsy disorder.

DOI: 10.1038/ncomms3410

Drug screening in Scn1a zebrash mutant identies clemizole as a potential Dravet syndrome treatment

Scott C. Baraban1,2, Matthew T. Dinday1 & Gabriela A. Hortopan1

1 Epilepsy Research Laboratory, Department of Neurological Surgery, University of California, San Francisco, Box 0520, 513 Parnassus Avenue San Francisco, California 94143, USA. 2 Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California 94143, USA. Correspondence and requests for materials should be addressed to S.C.B. (email: mailto:[email protected]

Web End [email protected] ).

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Epilepsy can be acquired as a result of an insult to the brain or genetic mutation. Among the genetic epilepsies, more than 650 variants have been identied in the SCN1A gene1,2.

Missense or frame-shift mutations in this gene are associated with generalized epilepsy with febrile seizures plus3 as well as a more severe disorder known as Dravet syndrome (DS). Children with DS initially exhibit normal development but often experience febrile seizure episodes within the rst year of life, with eventual progression to severe spontaneous recurrent seizures, intellectual disability, ataxia and psychomotor dysfunction. Seizures are inadequately managed using available antiepileptic drugs (AEDs) and these children are poor candidates for neurosurgical resection4.

In mammalian brain, there are four main subtypes of voltage-gated sodium channel a-subunits: NaV1.1, NaV1.2, NaV1.3 and

NaV1.6, encoded for by the genes SCN1A, SCN2A, SCN3A and SCN8A, respectively. Opening of these channels produces a sodium conductance and rapid cell membrane depolarization, for example, features integral to action potential initiation5. In mice, Nav1.1 is widely expressed in the central nervous system (CNS), including the axon initial segment of parvalbumin-positive hippocampal interneurons and excitatory principal cells6,7. Heterozygous deletion of Nav1.1 in mice leads to a reduction in the ring capability of acutely dissociated fast-spiking inter-neurons8. Mice with global or interneuron-specic heterozygous deletion of Nav1.1 exhibit temperature-induced and spontaneous seizures, mild ataxia, autism-like behaviours and premature death810. Knock-in mouse carrying a premature stop codon in domain III of the Nav1.1 channel also exhibit a decrement in spike amplitude during prolonged interneuron ring and increased sensitivity to temperature-induced seizures11.

Generation and characterization of valid animal models is critical to efforts to understand the pathophysiology of DS, and to aid in the identication of novel therapies. Although considerable attention has focused on modelling SCN1A mutations in mice, these animals have proven to be difcult to breed, and epilepsy phenotypes are strongly inuenced by the background strain genetics. Induced pluripotent stem cells can be generated from DS patients, but individual neurons do not recapitulate the network environment necessary for in-vivo seizure generation. Danio rerio (zebrash), a simple vertebrate species, provide an alternative model system with signicant advantages for genetic manipulation, cost-efcient breeding and in-vivo drug discovery1214. Ideally, an animal model should be based on a known genetic cause of the disease (SCN1A mutation), accurately recapitulate key features of the disease (epilepsy), and respond, or not, to therapies commonly used in patients with the disease (pharmacological validation). If successful, such a model could inform our understanding of the disease process and catalyse explorations towards new therapies. In zebrash, the voltage-gated sodium channel family consists of four sets of duplicated genes: scn1Laa and scn1Lab, scn4aa and scn4ab, scn5Laa and scn5Lab, and scn8aa and scn8ab (ref. 15). The zebrash scn1Lab gene shares a 77% identity with human SCN1A and is expressed in the CNS. A homozygous zebrash mutant for this gene (originally termed didys552) was discovered in a chemical mutagenesis screen using the optokinetic response as an assay16. These types of screens are based on inducing random point mutations using the alkylating agent N-ethyl-N-nitrosourea; the resulting mutations are typically loss-of-function and recessive. Although this is a homozygous mutation, scn1Lab zebrash mutants are relevant for the autosomal dominant human DS given the genome duplication in zebrash and the presence of an additional Nav1.1 homologue (scn1Laa). In this paper, we characterized scn1Lab mutants at the molecular and behavioural level, demonstrated that mutants exhibit spontaneous

drug-resistant seizures and then used them in a novel high-throughput screening programme to identify compounds that ameliorate the epilepsy phenotype. A phenotype-based screen identied clemizole, an US Food and Drug Administration (FDA)-approved compound, as an effective inhibitor of spontaneous convulsive behaviours and electrographic seizures in these mutants.

ResultsDevelopmental scn1Lab expression and characterization. Zebrash with a mutation in domain III of a voltage-gated sodium channel were identied by Dr Herwig Baier during a chemical mutagenesis screen16. We backcrossed original scn1Lab mutants onto the Tupfel long background for seven to ten generations and conrmed a methionine (M) to arginine (R) mutation in our colony (Fig. 1a). Reverse-transcriptase and quantitative (q) PCR revealed a decrease in mRNA expression for scn1Lab in mutant larvae at 3, 5 and 7 days post fertilization (dpf; Fig. 1b); antibodies recognizing this protein in zebrash are not available. As expected15, scn1Lab is prominently expressed during early stages of larval development (Fig. 1b) and specically in the CNS at 3 dpf (Fig. 1d,e). Whole-mount in situ hybridization revealed diffuse but prominent expression in the brain regions corresponding to the forebrain (telencephalon), optic tectum and cerebellum. A similar expression pattern was observed for scn1Laa at 3 dpf. At 5 and 7 dpf, CNS expression remained prominent and faint scn1Lab signal was also noted in the heart (Fig. 1d). Relative expression of scn8aa or scn8ab (Nav1.6), for example, a subunit thought to act as a genetic modier of DS17, failed to reveal a signicant difference in the expression between mutants and sibling controls at 5 dpf (Fig. 1c). Similarly, microarray analysis at 5 dpf also failed to detect a compensatory change in the mRNA expression of 13 different zebrash scn subunits (Table I), including the other homologue (scn1Laa). These results demonstrate a selective defect in a zebrash Nav1.1 gene expressed in the CNS during early development.

Large-scale transcriptomic analysis of scn1Lab mutants. Although inherited disorders of voltage-gated ion channels are recognized as an aetiology of epilepsy, investigation of transcriptional changes has not been reported for any epilepsy-related channelopathy. To detect differences in gene expression in an unbiased manner, we used an Agilent Danio rerio chip covering B44,000 probes (Fig. 2a,b). Hierarchical clustering analyses showed that B2.5% (1,099) of these probes (see Supplementary Data 1) were differentially expressed between mutants and sibling controls at 5 dpf (Pr0.01, Students t-test; 674 upregulated and 425 downregulated); 405 were assigned to an unknown function category. A list of 30 down- and upregulated known genes showing the greatest differences in expression is shown in Fig. 2c. Surprisingly, these differences were modest as 90% (990/1,099) of the identied genes exhibited fold-changes between 0.8 and 2.0. Similar to microarray analysis of Mecp2 single-gene mutant mice18, many of the genes identied had no obvious CNS-related function and/or expression. The two largest fold-changed genes, somatolactin b, and a Na, K-ATPase, have expression primarily restricted to the pituitary (smtlb)19 or the ear, intestinal bulb and pronephric duct (atp1a1a.5)20. Probes for several genes related to apoptosis (casp8, casp8b and casp3b) did not reveal any statistically signicant changes in the microarray studies. Of the genes with altered expression in scn1Lab mutants, six were previously implicated in neurological disorders, for example, pcdh19 (infantile epileptic encephalopathy), cyp1 and fxr2 (Fragile X syndrome), ocrl (Lowe syndrome), ubap2l (Parkinsons disease) and oca2 (Angelman syndrome). Microarray-based gene expression

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Figure 1 | Molecular characterization of scn1Lab zebrash mutants. (a) Sequencing conrmed a TG mutation in scn1Lab mutant complementary DNA. (b) Verication of reduced expression in scn1Lab mutants compared with sibling controls (Sib Ctrl) at 3, 5 and 7 dpf using qPCR. Data presented as means.e.m.; *signicance taken as Po0.05 Students t-test. Data were normalized to the internal reference gene b-actin. Values represent averages from ve independent biological samples (one sample ten pooled larvae) for each of the three developmental stages. Data presented as means.e.m.;

*signicance taken as Po0.05 Students t-test. (c) Relative expression of scn8aa and scn8ab in Nav1.1 mutants (n 5) and sibling controls (n 5) at 5 dpf.

Data presented as in b. (d) Whole-mount in situ hybridization for scn1Lab in larval zebrash at 3, 5 and 7 dpf. Wild-type larvae are shown in lateral views; expression is shown in dark purple. Scn1Laa expression at 3 dpf is shown for comparison. Heart indicated by arrowheads in 5 and 7 dpf panels. (e) Dorsal view of scb1Laa expression at 3 dpf; note the prominent expression in regions corresponding to the larval zebrash CNS. Cb, cerebellum; Tel, telencephalon; TeO, optic tectum; Scale bars, 0.35 mm in d, 0.2 mm in e.

Table 1 | scn channel subunit expression on microarray.

Accession Gene Description Fold M NM_200132 scn1Laa Danio rerio sodium channel, voltage-gated, type I, alpha, mRNA 0.919 0.12

NM_001077539 scn1ba Danio rerio sodium channel, voltage-gated, type I, beta a, mRNA 1.293 0.37 NM_001128156 scn1bb Danio rerio sodium channel, voltage-gated, type I, beta b, mRNA 1.004 0.01 NM_001077629 scn2b Danio rerio sodium channel, voltage-gated, type II, beta, mRNA 1.032 0.05 NM_001080802 scn3b Danio rerio sodium channel, voltage-gated, type III, beta, mRNA 1.115 0.16 NM_001039825 scn4aa Danio rerio sodium channel, voltage-gated, type IV, alpha a, mRNA 1.063 0.09 NM_001045065 scn4ab Danio rerio sodium channel, voltage-gated, type IV, alpha b, mRNA 0.738 0.44

NM_001077570 scn4ba Danio rerio sodium channel, voltage-gated, type IV, beta a, mRNA 1.115 0.16 NM_001077573 scn4bb Danio rerio sodium channel, voltage-gated, type IV, beta b, mRNA 0.956 0.06

NM_131628 scn8aa Danio rerio sodium channel, voltage-gated, type VIII, alpha a, mRNA 1.351 0.43 NM_001045183 scn8ab Danio rerio sodium channel, voltage-gated, type VIII, alpha b, mRNA 1.246 0.32 NM_001044922 scn12aa Danio rerio sodium channel, voltage gated, type XII, alpha a, mRNA 1.123 0.17 NM_001045123 scn12ab Danio rerio sodium channel, voltage gated, type XII, alpha b, mRNA 1.003 0.00

measurements were veried for 14 randomly selected genes using qPCR (Fig. 3a). Biological functions were assigned to all genes using gene ontology annotations, and the 482 genes showing at least a 1.5-fold change in expression and a P value o0.01 were categorized further (Fig. 3c). Calcium ion binding genes include annexinA1c, A1b and 2a, spectrin a2, neurexin 2a, calsyntenin 1 and parvalbumin 3. Signicant changes in a gap junction channel (cx43), a gene involved in clustering of voltage-gated sodium channels at the axon initial segment (spna2) and the ubiquitin domain of a GABA receptor (map1lc3b) were also noted. Three additional genes not found on the microarray were chosen for qPCR analysis (Fig. 3b): hcn1, a gene shown to be correlated with SCN1A using data mining (Starnet2; http://vanburenlab.medicine.tamhsc.edu/starnet2.shtml

Web End =http://

http://vanburenlab.medicine.tamhsc.edu/starnet2.shtml

Web End =vanburenlab.medicine.tamhsc.edu/starnet2.shtml ) and down-regulated in several seizure models21 was signicantly reduced in scn1Lab mutants compared with that in sibling control (Po0.05, two-tailed Students t-test). However, genes involved in synaptogenesis related to the formation of recurrent excitatory synapses and epilepsy22,23, for example, homer and bdnf, were unchanged.

Spontaneous seizures in scn1Lab mutant zebrash. Next, we monitored scn1Lab mutants for evidence of spontaneous electrographic seizures starting at 3 dpf, for example, the rst larval stage at which epileptiform discharge can be detected2428.

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Figure 2 | Microarray analysis of scn1Lab zebrash mutants. (a) Heat maps depicting the expression of genes differentially expressed between scn1Lab mutant and sibling control (Sib Ctrl) larvae at 5 dpf. Rows represent individual genes. Columns represent different larvae. Genes that are highly expressed in scn1Lab mutants relative to controls are shown in red. (b) MA plot of normalized microarray data for all 44,000 genes. The log-ratio M and the mean uorescence intensity A were calculated as the averages for all replicates. (c) A list of the top 30 genes showing the greatest differences (upregulated genes are shown in red and downregulated genes are shown in blue) in expression between scn1Lab mutants and Sib Ctrls.

Mutant larvae were identied by their black appearance (Fig. 4a), which is indicative of a defect in pigment aggregation and die prematurely between 10 and 12 dpf, as reported previously15. The forebrain extracellular eld recordings from paralysed and agar-immobilized scn1Lab mutants were marked by frequent brief interictal-like bursts and large-amplitude, long-duration, ictal-like events starting at 3 dpf (n 4) and pro

gressively becoming more prominent between 4 and 7 dpf (n 132; Fig. 2c). These events were conrmed in 100% of

mutants at 3 dpf, 100% at 4 dpf, 97% at 5 dpf, 98% at 6 dpf and 100% at 7 dpf. Temporal expansions of seizure activity and comparisons with single action potential and twitch artefact recordings are shown in Supplementary Fig. S1. Abnormal electrical events were never observed in age-matched sibling controls at any developmental stage (n 36). Hyperthermia-induced

seizures26 could be evoked in 5 dpf scn1Lab mutants and controls at apparently similar temperature thresholds (mutant: 26.90.5 C; n 14; control: 25.90.5 C; n 14; p 0.164, Students

t-test). However, these measurements were complicated, in mutants, by simultaneous occurrence of high-frequency, spontaneous epileptiform discharges. Mutants had elevated levels of swim activity and exhibited unprovoked seizure-like behaviour consisting

of whole-body convulsions and rapid undirected movement starting at 4 dpf (n 36). A representative locomotion tracking

plot of a scn1Lab mutant showing hyperactivity and convulsive behaviour is shown in Fig. 4b. This behaviour is similar to that classied as a Stage III seizure in larvae exposed to pentylenetetrazole27. Seizure behaviours were never observed in controls at any stage of development (n 36). In pools of mutant and sibling

control larvae, scn1Lab mutants stay close to the sides of the petri dish, which is considered a form of thigmotaxis in sh29. These results reveal a striking epilepsy phenotype in scn1Lab mutant zebrash.

Pharmacological evaluation of scn1Lab mutant zebrash. Seizures associated with SCN1A mutations are poorly responsive to most AEDs. To evaluate pharmaco-sensitivity, we recorded spontaneous electrographic seizures in agar-embedded scn1Lab mutants (56 dpf) under baseline conditions, and again after application of a commercially available AED. All drugs were bath applied at a concentration of 1 mM; seven sh were tested for each drug. Epileptiform event frequency (including interictal- and ictal-like discharges) and the fractional time spent seizing in

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Figure 3 | Quantitative reverse-transcriptase PCR analysis of scn1Lab zebrash mutants. (a) Comparison of the gene expression fold-changes obtained by microarray analysis (array) and real-time qPCR analysis. The y axis represents the average fold change in gene expression of each gene from zebrash at 5 dpf. The x axis represents different genes. (b) qPCR analysis of three genes involved in epileptogenesis. The relative gene expression is presented as log2 ratios to the least abundant transcript (log2DDct). Data were normalized to the internal reference gene b-actin. Values represent averages from ve independent biological samples (one sample ten pooled larvae). Bars indicate s.e.m.; *Po0.05 Students t-test. (c) Gene ontology classication of

differentially expressed genes detected in scn1Lab mutants at 5 dpf (Po0.05, ANOVA one-way and fold-changes 41.5). Biological processes representing at least ve gene annotations in at least one category are displayed.

scn1Lab mutants were reduced by valproate, diazepam, potassium bromide and stiripentol (Fig. 5a,b,d). Burst durations were not signicantly changed for any of these drug exposures (Fig. 5c). As expected, most AEDs had no effect and epileptiform activity became more frequent following exposure to carbamazepine (in two out of seven sh), ethosuximide (four out of seven sh) or vigabatrin (six of seven sh). As DS children often respond to the ketogenic diet (KD)30, we exposed a separate clutch of scn1Lab mutants, siblings and wild-type (WT) controls to a form of the diet31 for 48 h starting at 4 dpf. Locomotion tracking data on KD-exposed larvae at 6 dpf conrm a reduction in seizure-like behaviour to control levels in seven out of ten mutants (Fig. 5e; mean velocity, treated mutants 0.430.09 mm s 1, n 16;

untreated mutants 0.810.05 mm s 1, n 28; Po0.05,

KruskalWallis analysis of variance (ANOVA) on Ranks with a Dunns pairwise multiple comparison). No signicant differences in swim behaviour were noted in sibling controls treated with the KD (mean velocity 0.630.05 mm s 1, n 20) compared with

untreated WT larvae at 6 dpf (mean velocity 0.620.07

mm s 1; n 20). Acute exposure (20 min) to the diet had no

effect on mutant seizure behaviour in the locomotion assay (n 14; change in mean velocity o34%). Subsequent forebrain

eld recordings obtained from the same zebrash used in the locomotion assay (Fig. 5f, top trace) conrmed the occurrence of spontaneous epileptiform discharge for embryo media exposed scn1Lab mutants and a suppression of burst activity in mutants exposed to the KD for 48 h (Fig. 5f, bottom trace). These results demonstrate that the pharmacological prole for scn1Lab mutants resembles that seen in children with DS.

High-throughput drug screening in scn1Lab mutants. As the behavioural seizure activity is easily and rapidly monitored using a locomotion tracking format24,25,27,28,3236 (Figs 4b and 5e), we designed a relatively high-throughput phenotype-based strategy to screen chemical libraries for compounds that reduce mutant behaviour to Stage 0 (very little swim activity) or Stage I (increased, but non-convulsive, swim activity), for example, behaviour equivalent to that seen in normal WT controls. Automated measurement of larval activity was achieved using EthoVision tracking software (Noldus Information Technology) and a high-speed camera. Previous studies conrmed that high-velocity movement Z20 mm s 1 correspond to paroxysmal seizure-like convulsions (Stage III)35,36. Using a 96-well format, we automatically tracked mutant swim activity at baseline, and then again after addition of a test compound (100 ml); each compound was tested on 612 individual larvae at 5 dpf. The change in mutant swim activity between two consecutive recording epochs in the embryo media was taken as baseline and is shown in Fig. 6a (n 28). On the basis of an s.d. of 17.3 for baseline recordings

associated simply with a solution exchange, we screened for compounds that inhibited movement (measured as a change in mean velocity) by Z34%. To validate this approach, we rst screened 11 AEDs and the KD using this assay. As expected from electrophysiological assays (Fig. 5), diazepam, potassium bromide, stiripentol, valproate and a 48-h exposure to KD effectively inhibited seizure behaviour in the locomotion-based assay (Fig. 6b); ganaxolone, a neuroactive steroid related to allopregnalone, was also effective. Next, we screened test compounds at an initial concentration of 667 mM from a library that included

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a b

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Figure 4 | Spontaneous seizures in scn1Lab zebrash mutants. (a) Immobilized and agar-embedded zebrash larvae are shown. Images were obtained using a 4 objective and 2 magnier on an Olympus upright microscope during forebrain electrophysiological recordings in sibling control (Sib

Ctrl) (a, left) and scn1Lab mutant (a, middle) larvae at 5 dpf. Note the dark pigmentation for mutants. Recording electrodes can be seen in a (left and middle) and the approximate site of the recording electrode tip in the forebrain (red circle) is shown using a representative HuC:GFP-labelled larvaein a (right). Scale bar, 100 mm. (b) Sample locomotion tracking plot for Sib ctrl (b, left) and scn1Lab mutant (b, right) larvae at 5 dpf. (c) Representative 10 min recording epochs obtained in the forebrain of paralysed, immobilized and agar-embedded scn1Lab mutant larvae between 3 and 7 dpf. Note the presence of small and large amplitude spontaneous burst discharge; additional temporal expansions of seizure activity are shown in SupplementaryFig. S1. A representative recording, under identical recording conditions, from an Sin Ctrl larvae at 5 dpf is also shown. Scale bar, 2 mV; 30 s.

the US FDA-approved and toxicology-tested drugs (the International Drug Collection; http://www.msdiscovery.com/spectrum.html

Web End =http://www.msdiscovery.com/spec http://www.msdiscovery.com/spectrum.html

Web End =trum.html ). Among the 320 compounds screened in vivo, 18 were found to signicantly inhibit spontaneous seizures in scn1Lab mutants to levels comparable to Stage 0 or Stage I behaviour and/ or reduce mean swim velocity (red circles in Fig. 6c). These 18 compounds were then re-tested on a separate clutch of scn1Lab mutants at concentrations of 667, 67 and 6.7 mM. In the initial screen, 81 compounds were identied as lethal, that is, no visible heartbeat or movement in response to touch after a 30-min exposure (see Supplementary Table S1) and were re-evaluated at a dilution of 1:100; none of these advanced further. The drug library included a number of additional compounds with putative anticonvulsant properties (beclamide, aminohydroxybutyric acid and tiletamine) that were also ineffective in the 96-well locomotion assay at 667 mM. Fourteen of the re-tested compounds either failed to successfully inhibit seizure behaviour in a second clutch of scn1Lab mutants or only suppressed behaviour at the highest drug concentration. Next we selected 4 (out of 18) compounds that were effective in reducing seizure-induced swim activity and mean velocity at all 3 drug concentrations for further testing: zoxazolamine, clemizole HCl, clorgiline HCl and tolperisone HCl (Fig. 6d). Each of these compounds was evaluated a third time in the locomotion assay at a concentration of 100 mM, and subsequently monitored for forebrain electrographic activity. Clorgiline (a monoamine oxidase A inhibitor) and the muscle relaxants zoxazolamine37 and tolperisone38 were identied as false positives, because they reduced swim activity at this concentration, but when the same mutant was embedded in agar electrographic seizure events were still observed (see Fig. 6e). Only one compound, clemizole (antihistamine and NS4B RNA binding inhibitor)39,40, was effective in suppressing spontaneous

seizure activity in both assays (Fig. 6d,e). A comparison of elec

trophysiological burst characteristics for untreated (n 3) and

clemizole-treated (n 7) scn1Lab mutants shown in Fig. 6e

zebrash indicates a statistically signicant suppression of activity (untreated: burst frequency 1.50.3 bursts min 1; burst duration 926414 ms; fractional time spent seizing 0.730.17%;

versus clemizole: burst frequency 0.20.01 bursts min 1;

burst duration 154127 ms; fractional time spent seizing

0.030.02%; P 0.001 for all comparisons, KruskalWallis

ANOVA with a Dunns pairwise multiple comparison test). Clemizole had no signicant effect on seizure behaviour in the locomotion assay at concentrations between 6.25 and 50 mM (n 33). As an additional evaluation of the therapeutic potential

for acute clemizole treatment, we also demonstrated that 100 mM clemizole was effective in reducing seizure behaviour in WT zebrash exposed to 15 mM pentylenetetrazole (Fig. 6d; n 10),

that is, a model of acute seizures based on GABA receptor antagonism. These results suggest that scn1Lab mutants can be used in a high-throughput screen to identify potential lead compounds for DS.

DiscussionThe scn1Lab zebrash mutant described here is the rst simple vertebrate model of a sodium channel mutation that recapitulates features of DS, a catastrophic form of drug-resistant epilepsy in children. We have shown that these mutants exhibit hyperactivity, including convulsive behaviour, spontaneous electrographic seizures, shortened lifespan and a pharmacological prole similar to the human condition. Additional molecular analysis of scn1Lab mutants suggests the absence of gross changes in global gene expression and a lack of compensation, at the RNA level, by other

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a b

Acetazolamide Carbamazepine

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Figure 5 | Pharmacological validation of scn1Lab zebrash mutants. (a) Heat map showing the response to nine different AEDs. Each column represents the per cent change in burst frequency (baselinedrug/baseline 100) for one individual zebrash mutant. Drugs that inhibit seizure events

are shown in dark blue. All drugs were tested at a concentration of 1 mM. Note in some trials, carbamazepine and vigabatrin increased burst frequency over the initial baseline levels. (b) Plot of the mean change in burst frequency and s.e. for the data shown in the heat map. Paired t-test or Wilcoxon signed-rank sum test for data that failed the normality test showed signicance as follows: diazepam (P 0.002; n 7), potassium bromide (P 0.016; n 7),

stiripentol (P 0.024; n 7) and valproate (P 0.004; n 7). (c) Plot of the burst duration for all trials shown in a. Data are presented as the

means.e.m. for electrographic seizure events at baseline (black bars) and after drug exposure (white bars). Inset shows a representative 2-min recording during the stiripentol trial; scale bars, large trace 1 mV, 1 s; small trace 1 mV, 100 ms. (d) Plot of the fractional time spent seizing for all trials shown in a. Data are presented as the means.e.m. for electrographic seizure events at baseline (black bars) and after drug exposure (white bars). Students t-test or MannWhitneyRank sum test for data that failed the normality test showed signicance as follows: diazepam (P 0.001; n 7); potassium bromide

(P 0.043; n 7); stiripentol (P 0.007; n 7) and valproate (P 0.007; n 7). (e) Locomotion tracking plots for ten individual mutant larvae raised in

embryo media (top row) or the KD for 48 h. Plots show swim velocity and locomotion tracks with darker colours indicative of higher velocities; 10-min trials are shown. (f) Representative 10-min extracellular recording epochs from the same sh shown in e; representative examples are indicated by * in the locomotion plots. Scale bar, 1 mV, 30 s. Inset shows burst at higher temporal resolution (indicated by #); scale bar, 1 mV, 100 ms.

voltage-gated Na channel subunits. A two-stage phenotype-based drug screening strategy to identify lead compounds with the potential to ameliorate epilepsy phenotypes associated with SCN1A mutation identied one FDA-approved drug (clemizole).

Electroencephalographic activity is typically normal in the rst year of life for DS patients, with an evolution to abnormal paroxysmal and polyspike activity between 1 and 9 years of age. This age-dependent pattern was mimicked in the developing zebrash larvae at ages where scn1a expression was signicant. The forebrain extracellular recordings in very young larvae (3 dpf) appeared largely normal with the occasional small burst of polyspike activity. Frequent brief interictal-like activity with large amplitude polyspike burst discharges became more prominent as larvae aged. The architecture of these electrical events resembled those previously described in WT larvae exposed to pentylenetetrazole27, 4-aminopyridine24, linopirdine28 or hyperthermia26.

The appearance of electrographic seizure activity corresponds with hyperactivity, full-body convulsions with associated high-velocity swim activity and brief loss-of-posture in freely behaving mutants. These types of spontaneous behaviours are never observed in WT larvae and, again, resemble those previously observed only during exposure to convulsant drugs. These behaviours are an indirect indicator of seizure activity and could be used for rapid in-vivo evaluation of drug treatments and lethality in a multi-well format using automated locomotion tracking software32,33,35. We also show that seizures in scn1Lab zebrash mutants are responsive to the KD and four AEDs (for example, valproate, benzodiazepine, potassium bromide and stiripentol) prescribed clinically for patients with DS41.

Interestingly, electrographic seizure events in scn1Lab mutants remained unchanged (or perhaps worsened) in response to several commercially available AEDs. Although it is possible that

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a b

% change from baseline

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scn1Lab mutant (untreated)

scn1Lab mutant (Clem)

scn1Lab mutant (Zox)

**

*

** ** **

Figure 6 | Screen to identify drugs that rescue scn1Lab mutant epilepsy phenotype. (a) Box plot of mean velocity (in mm s 1) for two consecutive recordings of mutant larvae in embryo media. The per cent change in velocity from baseline (recording 1) versus experimental (recording 2) is shown. The bottom and top of the box represent the 25th and 75th percentile, respectively. The horizontal line represents median value; vertical lines encompass the entire range of values. (b) Plot of the effect of AEDs on seizure behaviour in scn1Lab mutants. Bars represent per cent change in mean velocity; 612 sh per experiment. Drugs tested at 1 mM; diazepam (Dzp; Po0.001), carbamazepine (Carb; P 0.024), ganaxolone (Gan; P 0.003), stiripentol (Stp;

P 0.001), valproate (Vpa; P 0.026) and a 48-h KD (P 0.003) exposure reduced seizure activity, measured as a change in velocity, by more than

the s.d. in control recordings (34%, dotted line). Acetazolamide (Acet; Po0.001) and ethosuximide (Etx; P 0.250) increased seizure behaviour;

levetiracetam (Lev; P 0.243) and lamotrigine (Ltg; P 0.058) had no effect. (c) Plot for all 320 compounds tested. Coloured circles (red) represent

positive hits; compounds that decreased the activity by 100% were generally toxic; 612 sh per trial. Arrowhead; rst clemizole trial. (d) Plot of drug re-trials on separate mutant clutches; 100 mM per drug; ten sh per trial. Abbreviations: Clem, clemizole; Clem PTZ, clemizole 15 mM PTZ; Clorg,

clorgiline; Tolp, tolperisone; Zox, zoxazolamine. Effect of acute clemizole on PTZ-induced seizures shown for WT larvae. Bars represent means.e.m. For panels b and d: Students paired t-test or MannWhitneyRank sum test with signicance set at *P 0.01 or **Po0.001. (e) Sample electrophysiology

recordings from scn1Lab mutants exposed to clemizole in the locomotion assay (d) and then monitored using a forebrain recording (top trace; ictal-like burst shown in inset). Similar traces for an untreated mutant (middle) and mutant treated with zoxazolamine (bottom). Scale bars, large traces0.5 mV, 10 s; inset 0.5 mV, 100 ms.

drug concentrations higher than 1 mM could be required to abolish electrical events, these would be considered high and potentially non-selective concentrations. In drug trials using an acute pentylenetetrazole (PTZ)-induced seizure model in larval zebrash24,32,33,42, AED concentrations of 1 mM and below were often sufcient for assessing antiepileptic activity. With a failure to respond to ten different AEDs, this model ts the clinical denition of drug-resistant epilepsy43. For nearly 40 years, the discovery and identication of new AEDs has almost entirely been based upon preclinical animal models of acquired or acute

seizures in rodents44. This approach successfully identied drugs that block generalized tonicclonic seizures in humans45 but remains time-consuming, resource intensive, expensive and laborious. Although testing against PTZ or other types of acquired seizures in zebrash larvae may be more efcient than similar assays in rodents32,33,42, they ultimately should identify the same classes of compounds. In contrast, here we describe an alternative screening strategy using a 96-well format for rapid automated behavioural monitoring followed by a sensitive electrophysiological assay of spontaneous electrographic seizure

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activity in a mutant sh mimicking a known human genetic disorder. Our in-vivo strategy simultaneously monitors lethality and is not limited to SCN1A, but could be applied to any monogenic epilepsy disorder. Indeed, this phenotype-based approach could form the basis of a genetically informed or personalized approach to drug discovery. Although genetically modied mice mimicking known SCN1A mutations and exhibiting epilepsy have been developed, breeding can be complicated, background strain can modify seizure phenotypes and AEDs are rarely tested in these animals. For example, in Scn1aRX/ mutant mice, stiripentol and clobazam were only evaluated for effects on hyperthermia-induced seizure thresholds46. Treatment of Scn1a / mutant mice with clonazepam, an allosteric modulator of GABA-A receptors, rescued some of the autistic-like behaviours but was not evaluated as an antiepileptic43. Where drug-resistant rodent epilepsy models have been described, such as the subgroup of WT rats selected from kindling or post-status epilepticus models47, they remain only poorly characterized and are not suitable to initial high-throughput stages of drug screening. In contrast, using a zebrash scn1Lab mutant with greater than 75% sequence identity for a human sodium channel mutation, we completed a large-scale transcriptomic proling of over 44,000 probes, demonstrated a developmental progression of scn1Lab channel expression and epileptic phenotypes, analysed the effects of available antiepileptic therapies and screened a 320-compound chemical library against spontaneous unprovoked seizures. Although this rst proof-of-principle screen was accomplished at 1 sh per well, 612 sh per trial and 1 trial per week, the ease with which zebrash could be scaled upward (especially in a commercial setting) to study 100s to 1,000s of larvae per week make this an attractive system for a rapid large-scale, rst-stage in-vivo drug discovery programme. Simultaneous in-vivo evaluation of toxicityone of the greatest sources of failure in moving lead compounds from the bench to the clinicis a critical advantage of our approach over available organotypic hippo-campal culture- or in silica-based screening strategies.

Although any animal model drug discovery data should be treated cautiously, clemizole, a compound with H1 antagonist and NS4B RNA-inhibiting properties39,40, is an FDA-approved drug with a safe toxicology prole emerged from this screen and offers an exciting starting point for further research. For example, although it was recently recognized that antihistamines inhibit induced seizures in neonatal rats48, this may not be the mechanism of action here, as four other H1 antihistamines (pimethixene maleate, chloropyramine HCl, mebhydrolin napthalenesulphonate and iproheptine) failed to suppress convulsive behaviour in scn1Lab mutants. Furthermore, evidence suggests the potential for H1 antihistamines to adversely modify seizures in children49, indicating that a more detailed analysis will be required to identify a mechanism of action. Given that clemizole was also effective in a zebrash version of the Metrazol test, it may be worthwhile to pursue additional preclinical testing in the NIH-sponsored Anticonvulsant Drug Development Program at the University of Utah. Most importantly, our studies suggest that in-vivo drug screening and experimental analysis of scn1Lab mutant zebrash could prove extremely valuable to the understanding (and treatment) of DS.

Methods

Animals. Scn1Lab (didys552) zebrash embryos were a kind gift from Herwig Baier. Adult HuC:GFP zebrash were a kind gift from Stephen Ekker. Zebrash were generated and maintained in accordance with the guidelines of the University of California, San Francisco, Committee on the Use and Care of Animals. Zebrash larvae were maintained in embryo medium consisting of 0.03% Instant Ocean (Aquarium Systems, Inc., Mentor, OH, USA) in deionized water containing 0.002% Methylene Blue as a fungicide. Larval zebrash clutches were bred from scn1Lab

heterozygous animals that had been backcrossed to Tupfel long WT or HuC:GFP zebrash for at least seven generations. Homozygous mutants (sorted based on pigmentation) and age-matched sibling larvae were used. Although the precise genetic defect responsible for the skin pigmentation issue is unknown, it is interesting that a 1.5-fold upregulation of a gene encoding the melanocortin 5a receptor was noted in our microarray data.

Seizure monitoring. Procedures for locomotion tracking and electrophysiology were described24,27. In pilot experiments, HuC:GFP zebrash were used in electrophysiology experiments to obtain an estimation of the location of recording electrodes. Locomotion plots were obtained for one sh per well at a recording epoch of 10 min using a DanioVision system running EthoVision XT software (Noldus Information Technology, Leesburg, VA, USA). Seizure scoring was performed as described27. Locomotion plots were analysed for distance travelled (in mm) and mean velocity (in mm s 1). Epileptiform events were analysed in pClamp (Molecular Devices, Sunnyvale, CA, USA) and dened as upward or downward membrane deections greater than 2 baseline noise level, and were

classied as interictal-like (100 to 300 ms duration) or ictal-like (1,000 to 5,000 ms duration). Burst frequency was determined by counting the number of epileptiform events per minute during a 10-min recording epoch. Burst duration was determined by measuring the onset-to-offset interval for all events during the same epoch.

Drugs. Drugs were obtained from Sigma-Aldrich and were dissolved in embryo media. Stock solutions were prepared in embryo media at 1 mM and pH adjusted to B7.5. Ganaxolone was a kind gift from BioCrea GmbH (Radebeul, Germany).

Compounds for drug screening were purchased from MicroSource Discovery Systems, Inc. (International Drug Collection, Gaylordsville, CT, USA) and were provided as 10 mM dimethylsulphoxide (DMSO) solutions. Test compounds were dissolved in embryo media and tested at concentrations between 6.7 and 667 mM;

nal DMSO concentration B7%. An initial screen concentration of 667 mM was chosen for behavioural studies in freely swimming sh, as this falls on the lower range of AED concentrations previously reported in to be effective against PTZ (1020 mM)-induced seizures in larval zebrash (0.125 mM)27,32,42 and was the most efcient use of the small volume of stock solution (250 ml) provided by

MicroSource Discovery Systems, Inc. A slightly higher concentration (1 mM) was chosen for the initial AED validation assays in Figs 5 and 6 to account for any potential complications associated with diffusion through the agar. DMSO was evaluated for toxicity at dilutions between 0.01 and 100% using WT larvae (n 12

sh per concentration); DMSO at 425% was lethal. In all drug-screening studies compounds were coded and experiments were performed by investigators blind to the nature of the compound. Baseline recordings of seizure activity were obtained from mutants bathed in embryo media; a second plot was then obtained following a solution change to a test compound. Each test compound classied as a positive hit in the locomotion assay was visually conrmed as alive based on the movement in response to touch and visible heartbeat. WT sh exhibit little to no spontaneous swim activity during these 10-min recording epochs (see Fig. 3b) and were not used in the drug-discovery assay.

Molecular biology. Procedures for microarray, qPCR and whole-mount in-situ hybridization were described25. See Supplementary Tables S2 and S3 for primer sequence data.

Data analysis. Data are presented as mean and s.e.m., unless stated otherwise. Pairwise statistical signicance was determined with Students two-tailed unpaired t-test, ANOVA or MannWhitneyRank sum test, as appropriate, unless stated otherwise. Results were considered signicant at Po0.05, unless otherwise indicated.

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Acknowledgements

We thank Herwig Baier and Stephen Ekker for zebrash lines used in this study; the Sandler Asthma Basic Research (SABRE) Center Functional Genomics Core Facility and NIH/NCRR UCSF-CTSI Grant Number UL1 RR024131 for the microarray support; Wyatt Potter, Robert Hunt and MacKenzie Howard for critical comments on the manuscript; and Rosanne Estrada for assistance with qPCR. This work was supported by a EUREKA grant from the NINDS (1 R01 NS079214), a Research Award from the Dravet Syndrome Foundation (http://www.dravetfoundation.org/

Web End =http://www.dravetfoundation.org/) and a Challenge Award from Citizens United for Research in Epilepsy (http://www.cureepilepsy.org/home.asp

Web End =http://www.cureepilepsy.org/home.asp) to S.C.B.

Author contributions

S.C.B. designed the experiments, performed all electrophysiology studies, developed and screened compounds using the locomotion assay and wrote the manuscript; M.T.D. maintained the zebrash colonies and assisted in the drug screening assays; G.A.H. performed all the molecular biology experiments and data analyses.

Additional information

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How to cite this article: Baraban, S.C. et al. Drug screening in Scn1a zebrash mutant identies clemizole as a potential Dravet syndrome treatment. Nat. Commun. 4:2410 doi: 10.1038/ncomms3410 (2013).

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