Fishing for innovative alternatives for modeling epilepsy

Why zebrafish can pave the way for investigating epilepsy’s etiology and screening new anti-seizure drugs

By Carles Cornet, Project Manager at ZeClinics.

Carles Cornet is an expert in zebrafish biology and drug discovery. He did his master’s degree in biomedicine straddling between the University of Barcelona and the German Cancer Research Center in Heidelberg. Carles joined ZeClinics team in 2015 as a Ph.D. student, to work on the optimization of the zebrafish model as a novel tool to assess drug safety and antitumoral efficacy. After obtaining his Ph.D. in Biomedicine from the Pompeu Fabra University in Barcelona, in January 2020 he became a project manager of the company. He is currently involved in the toxicology and oncology areas, as well as in the CNS behavioral efficacy area.

In our previous blog article, we discussed the differences between in vitro, in vivo, and in silico assays in preclinical research and how zebrafish bridge between in vitro and in vivo models. In this new blog, we will address how zebrafish can help in epilepsy research.

The epidemiology of global epilepsy

Epilepsy is the fourth most common neurological disorder affecting more than 50 million people worldwide, with the highest incidence present in young children and in the elderly. Its etiology is highly diverse and comprises environmental and genetic causes, with over 140 associated genes identified to date [1].  Epilepsy is a life-shortening disease where the risk of premature death is up to three times higher than for the general population. It is characterized by spontaneous and abnormal synchronous neuronal hyperactivity, which can result in different symptoms such as unprovoked recurrent seizures, loss of consciousness, disturbances in movement and sensation (including vision, hearing, and taste), and mood (anxiety, depression) as well as other cognitive functions.

Despite significant progress in understanding the molecular mechanisms of epileptogenesis, as well as the introduction of over 20 new antiseizure drugs since 1993, approximately one-third of patients remain resistant to currently available treatment options. This is partly explained by the fact that most of the current treatments act at the level of suppressing the seizures, rather than treating the underlying mechanisms.

Human neurological and genetic tests are crucial to unraveling the etiology of this neurological disorder. However, the ethical issues, the lack of sample numbers, and the need for proper controls (many patients have already been exposed to anti-epileptic drugs) in the human clinical studies highlight the need for complementary models. Mammalian models of epilepsy have contributed to gathering knowledge on the mechanisms underlying epilepsy, but the high costs of breeding, low throughput, and regulatory limitations in their experimentation reduce their utility in drug screens. Zebrafish can help in the investigation of epilepsy’s etiology, as well as being suitable for in vivo drug screenings.

Zebrafish as a model for epilepsy research

Zebrafish have several experimental advantages such as small size, large number of progenies that develop externally, fast life cycle, fully sequenced genome, high genetic and physiological homology with humans, and ease of genetic manipulation, which make zebrafish a highly robust model for understanding human disease and to be used in large-scale approaches.

More relevant to neurological disorders are its rapid neurodevelopment and its similitude with mammals in the main central nervous system subdivisions, i.e. forebrain, midbrain, hindbrain, and spinal cord, and the common neurotransmitter system, i.e. γ–aminobutyric acid (GABA), glutamate, dopamine, norepinephrine, serotonin, and acetylcholine.

Figure 1: Anatomical and functional similarities between zebrafish and human brains.

Finally, other important advantages of zebrafish for epilepsy research include: 

  1. Its ever-expanding genetic toolbox [2], which allows to perturb and monitor neural or glial activity in the entire brain and thus, model human genetic forms of epilepsy.
  2. The possibility to perform electroencephalographic recordings in both larval and adult fish.
  3. The high throughput behavioral analysis using automated video tracking systems. 

Altogether, the zebrafish model offers enormous opportunities to understand the genetic and environmental causes of epilepsy and can help in the discovery of novel antiseizure and epilepsy therapeutic targets and therapies.

Figure 2: Imaging technique capturing the activity of an entire zebrafish brain, at single-cell resolution. Light-sheet microscopy to record activity, reported through the genetically encoded calcium indicator GCaMP5G, from the entire volume of the brain of the larval zebrafish in vivo at 0.8 Hz, capturing more than 80% of all neurons at single-cell resolution. Obtained from: Misha B. Ahrens, et al., 2013.

Zebrafish PTZ model

Several pharmacological and genetic epilepsy models have been generated in zebrafish, nicely reviewed in [3]. Among the pharmacological models of epilepsy, the most well known and more commonly used model is the one using Pentylenetetrazole (PTZ). PTZ is a well-established convulsant drug, which acts as an antagonist of the GABA(A) receptor, increasing the closed state of the channel and therefore enhancing overall neuronal excitation. PTZ has been used since late 1960s in rodents for antiseizure drug discovery and is generally predictive for compounds with activity against generalized absence and myoclonic seizures in humans [4]. In zebrafish, it was firstly used 17 years ago and demonstrated that PTZ produces similar behavioural, electrophysiological and molecular alterations than rodent models [5]. Since then, the zebrafish PTZ model has been increasingly used in laboratories worldwide and has been shown to confirm the same antiepileptic drug candidates as the rodent PTZ models.

Figure 3: Videotracking of larval response to a seizure-inducing visual stimulus acquired by Daniovision™ device (Noldus IT). Larvae from the first two columns are treated with the negative control, while those from the last two columns are treated with PTZ. Red dot depicts the moment when the flash of light has occurred. Subsequently, PTZ-treated larvae begin to convulse.

Examples of success

However, perhaps the most successful example showing the power of the zebrafish model in epilepsy is the one concerning Dravet Syndrome (DS). DS is one of the most severe genetic pediatric epilepsies, producing severe intellectual disability, impaired social development, and persistent drug-resistant seizures. De novo loss-of-function mutations in the SCN1A gene are responsible for over 80% of DS cases. SCN1A gene codes for the α subunit of NaV1.1 voltage-gated sodium channel, which regulates the firing of neurons. In zebrafish, a loss-of-function mutation was identified in the scn1lab gene (zebrafish orthologue of the human SCN1A) after an ENU mutagenesis screen [6]. The scn1lab-/- zebrafish mutant was shown to have spontaneous recurrent seizures that responded favorably when treated with the DS “standard of care” therapies. However, similar to what occurs in patients with DS, they were pharmaco-resistant to most other antiepileptic drugs [6].

In the same study, a phenotype-based screen of 320 compounds revealed clemizole, an FDA-approved compound with anti-histaminic properties, as an effective inhibitor of spontaneous convulsive behaviors and electrographic seizures in scn1lab -/- mutant fish [6]. Since then, Clemizole (EPX-100) has satisfactorily passed through phase I for this new indication clinical study after showing a broad safety profile and is now under investigation as an ‘add-on treatment’ in a pivotal phase II clinical trial.

In posterior experiments, several other promising antiepileptic drugs have been identified such as fenfluramine (now FDA-approved as Fintepla®) [7], synthetic cannabinoids (similar to the FDA-approved cannabidiol Epidiolex®) [8], and the repurposed drugs (structure analogs of clemizole) trazodone (Desyrel®) and lorcaserin (Belviq®) [9]. Indeed, Lorcaserin has been successfully tested in a small, compassionate-use trial, significantly reducing the frequency and/or the severity of seizures in all five patients enrolled [9].

These are just a few success paradigms of larval zebrafish model usage for DS, many others are beautifully reviewed in [3]. Altogether, they demonstrate how rapid the path can be from preclinical discoveries in zebrafish, through target identification, to potential clinical treatments for DS.

ZeClinics - Epilepsy models

At ZeClinics we have behaviourally characterized and validated a pharmacological model of epilepsy (PTZ). This model allows for identifying compounds that prevent locomotor and convulsive behavioral alterations induced by PTZ. In addition, it can be used to discard molecules, at the early stages of the drug discovery pipelines, that induce PTZ-like seizures as potential proconvulsant drugs.

PTZ Epilepsy Model

High Throughput In Vivo Efficacy Screening Based On Complex Behavioral Endpoints

Furthermore, ZeClinics is an officially licensed CRISPR/Cas9 solutions provider under the Broad Institute of MIT Harvard. Hence, we can use zebrafish for performing target discovery, generate custom epilepsy genetic models, in order to obtain stable genetically-modified zebrafish lines, or transiently modify gene expression lines, if the biological interest resides mainly in evaluating the effect of gene loss-of-function or gene overexpression.

ZeGenesis Platform

Custom Gene-Edited Zebrafish From An Officially Licensed CRISPR Solutions Provider

Take-home messages

  • There is high conservation of convulsive behavioral responses between zebrafish and mammals.
  • The combination of zebrafish’s transparency and rapid external development with the vast repertoire of different cell/tissue reporter lines offers the possibility to study the mechanisms of epilepsy onset, and the effects of pharmacological intervention through high-resolution spatial and temporal imaging in an in vivo context.
  • Feasibility to perform high throughput antiseizure drug screens using zebrafish larvae. In vitro inputs with in vivo complex outputs.
  • Ease to genetically modify zebrafish generating loss-of-function/gain-of-function mutations to study the role of specific genes in the context of epilepsy. 

REFERENCES

[1] Ellis CA, Petrovski S, Berkovic SF. Epilepsy genetics: clinical impacts and biological insights. Lancet Neurol. 2020 Jan;19(1):93-100. https://doi.org/10.1016/S1474-4422(19)30269-8

[2] Cornet C, Di Donato V, Terriente J. Combining Zebrafish and CRISPR/Cas9: Toward a More Efficient Drug Discovery Pipeline. Front Pharmacol. 2018 Jul 3;9:703. https://doi.org/10.3389/fphar.2018.00703

[3] Gawel K, Langlois M, Martins T, van der Ent W, Tiraboschi E, Jacmin M, Crawford AD, Esguerra CV. Seizing the moment: Zebrafish epilepsy models. Neurosci Biobehav Rev. 2020 Sep;116:1-20. https://doi.org/10.1016/j.neubiorev.2020.06.010

[4] Porter RJ. Antiepileptic drug development program. Prog Clin Biol Res. 1983;127:53-66. https://pubmed.ncbi.nlm.nih.gov/6351101/

[5] Baraban SC, Taylor MR, Castro PA, Baier H. Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience. 2005;131(3):759-68. https://doi.org/10.1016/j.neuroscience.2004.11.031

[6] Baraban SC, Dinday MT, Hortopan GA. Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment. Nat Commun. 2013;4:2410. https://doi.org/10.1038/ncomms3410

[7] Dinday MT, Baraban SC. Large-Scale Phenotype-Based Antiepileptic Drug Screening in a Zebrafish Model of Dravet Syndrome. eNeuro. 2015 Aug 31;2(4):ENEURO.0068-15.2015. https://doi.org/10.1523/ENEURO.0068-15.2015

[8] Griffin A, Anvar M, Hamling K, Baraban SC. Phenotype-Based Screening of Synthetic Cannabinoids in a Dravet Syndrome Zebrafish Model. Front Pharmacol. 2020 Apr 24;11:464. https://doi.org/10.3389/fphar.2020.00464

[9] Griffin A, Hamling KR, Knupp K, Hong S, Lee LP, Baraban SC. Clemizole and modulators of serotonin signalling suppress seizures in Dravet syndrome. Brain. 2017 Mar 1;140(3):669-683. https://doi.org/10.1093/brain/aww342

CNSDisease modelingDisease modelsdrug discoveryepilepsyHigh Throughput Screeningneurologyphenotypic screeningpreclinical researchR&Dtarget validationZebrafish