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HomeInVivo Biosystems BlogDisease ModelingAn Updated Comparison of Common Model Organisms Part 2: Organisms for Modeling Epilepsy

An Updated Comparison of Common Model Organisms Part 2: Organisms for Modeling Epilepsy

Summary

According to the Epilepsy Foundation 1 in 26 people in the U.S.A will develop epilepsy at some point in their life, however, epilepsy is a very diverse condition which makes researching it especially challenging. In this blog, we will discuss the model organisms commonly used in epilepsy research, and how their characteristics act as advantages and limitations when modeling epilepsy. 

Introduction

Epilepsy is one of the most common neurological conditions worldwide, largely occurring in very old and very young people (Beghi and Giussani, 2018). Epilepsy is characterized by unpredictable seizures, but is a spectrum condition with a wide range of seizure types and causes including brain injury, tumors and inherited single-gene mutations (Epilepsy Foundation of America, 2020). For children with epilepsy, these seizures are particularly detrimental. As most children with epilepsy develop the condition before the age of two, their development is severely impacted; their seizures can lead to cognitive impairments and death (Beghi and Giussani, 2018; Holmes, 2016). Because of this, there is great interest and urgency in researching pediatric epilepsy. 

Understandably, research into the generation of a seizure, and the alterations that are seen in the brain network after a seizure, cannot ethically be conducted on humans. Thus, animal models are used to replicate epileptic seizures. Across the field of biomedical research animal models are used to identify compounds and therapeutics for human diseases which can then be tested in humans. This allows for faster, safer drug development. Recently, advancements in technology have enabled researchers to replace ortholog locus in animal models such as C. elegans, Zebrafish, and mice, with human cDNA. These genetically engineered animal models, known as humanized models, produce highly translatable results and have exciting implications for advancing personalized medicine (Aartsma-Rus & Putten, 2020).

The ability to have ‘custom made’ models to target specific genetic mutations is particularly helpful in epilepsy research because it is such a wide ranging condition (Aartsma-Rus & Putten, 2020). Over a hundred single gene causes of epilepsy have now been identified, with no one gene underlying more than 1% of total cases (Helbig & Ellis, 2020). Humanized models are just one tool used to investigate epilepsy, as, with all research, the procedure depends on the study being conducted. For instance, there has also been great success in identifying disease-causing mutations using knock-in technologies (Arief et al., 2018). 

Non-Mammalian organisms (Drosophila, Zebrafish, and C. elegans) have become the primary models for epilepsy, however, mammalian models (mice) are also used. We will discuss the advantages and limitations of these commonly used organisms for modeling epilepsy. 

Drosophila melanogaster (fruit fly)

As a model organism the Drosophila melanogaster has the advantages of a short generation time, small size, and few ethical concerns (Rosch et al., 2019). In epilepsy research the Drosophila has been widely adopted due to the similarity between its nervous system and the human nervous system. Notably for epilepsy research, Drosophila’s nervous system contain similar voltage-gated and ligand-gated signaling molecules to humans, like Na+, K+ and Ca2+ channels, as well as glutamate, acetylcholine and GABA transmitter receptors (Takai et al., 2020). Sodium channel blockers are used in therapy of epilepsy, and so, a Drosophila model provides a simple model that can perform large-scale screenings of antiepileptic drug candidates (Catterall, Kalume & Oakley, 2010). Furthermore, Drosophila are useful for behavioural analysis of epilepsy because their centralized brains are capable of producing complex behaviours such as seizure-like activity (Arief et al., 2018). 

Drosophila’s increasing popularity in epilepsy research makes it a particularly powerful model: its genome is completely sequenced, stock centers such as Bloomington Drosophila Stock Center have Drosophila mutant and RNAi lines readily available, and knock-in technologies using Drosophila are well-established (Takai et al., 2020; Arief et al., 2018). Knock-in technologies allow researchers to replace or disrupt a specific gene, and provide a powerful tool to identify disease-causing mutations. For example, mutant strains of Drosophila have been used to investigate the human SCN1A gene, which has over 600 possible mutations that have been associated with epilepsy syndromes (Arief et al., 2018; Catterall, 2012). 

Despite these advantages, there are drawbacks in working with Drosophila in epilepsy research. For instance, Drosophila have lower translatability to humans than other models because their overall anatomy is very different to humans (Arief et al., 2018). Also, while they exhibit complex behaviour this can be difficult to measure (Takai et al., 2020).

Caenorhabditis elegans (worm)

C. elegans have rapidly become one of the most commonly used model organisms because, unlike mammalian models, C. elegans are simplistic enough that researchers are able to follow, and gain insight, into how genes affect the development and behaviour (Arief et al., 2018). C. elegans are well-established as models of the nervous system thanks to their physiological similarities to humans: possessing ion channels, conserved neuron morphology, and the neurotransmitters glutamate, dopamine, serotonin, GABA and acetylcholine (Takai et al., 2020). 

C. elegans are also low cost, easily genetically modified, and carry few ethical concerns (Takai et al., 2020). There are national and global databases for the genome of C. elegans, which are continually updated as researchers use CRISPR/Cas9 gene editing to create loss-of-function mutations, and create ‘humanized worms’ by replacing a C. elegans protein with its human orthologue.’ These possibilities for genetic manipulation make C. elegans a powerful way to study the functional impact of specific genes on epilepsy expression. For example, mutations in STXBP1/Munc18‐1 protein are known to cause epileptic encephalopathy, and is increasingly associated with other forms of epilepsy and neurological diseases (Saitsu, et al. 2008; Zhu et al., 2020). Zhu and colleagues (2020) created humanized strains of C. elegans by replacing the worms’ UNC-18 protein with STXBP1, producing worms which expressed seizure-like activity.  At Invivo Biosystems we have created over 90 point mutations in the STXBP1 gene (see image 1), which allows for VUS (variants of Uncertain Significance) to be tested to see which are benign versus pathogenic. This ability to create humanized/targeted models is a significant advancement in personalized medicine.

96 Point Mutations in STXBP1

Image 1: A map of point mutations in the STXBP1 gene created via CRISPR

Unlike other organisms used for disease modeling, C. elegans don’t have voltage-gated sodium channels; this is a notable limitation for C. elegans when modelling epilepsy because mutations in sodium channels have been linked to genetic epilepsy syndromes, and sodium channel blockers are used for therapeutic treatment of seizures (Takai et al., 2020; Catterall, 2012). This being said, C. elegans offer an animal model which can be easily and effectively coupled with another model organism to reduce cost and time when studying epilepsy. 

Danio rerio (zebrafish)

Zebrafish are a highly used model in epilepsy research as they have a fully sequenced genome, and have transparent embryos which enables researchers to use in vivo imaging, such as calcium imaging and optogenetics, in their epilepsy studies. These imaging capabilities are valuable tools for studying the basic mechanisms underlying seizures. (Turrini et al., 2017). 

In recent years, Zebrafish has made a name for itself as a non-mammalian alternative to a mouse animal model because the macro-organization and cellular morphology of the Zebrafish brain is comparable to mice brains, while being a lower maintenance model than mice. (Takai et al., 2020). Also like mice, Zebrafish mutants of epilepsy-associated genes exhibit spontaneous seizures, making them useful models for behavioural analysis (Takai et al., 2020). 

Both adult and larvae Zebrafish can be used for studying epilepsy (Arief et al., 2018). As previously mentioned, Zebrafish larvae are transparent and easily genetically engineered; as a result, it is easy to monitor the development of these transgenic models’ nervous systems. Zebrafish larvae are used more than adults in epilepsy studies, however, adult Zebrafish have some notable benefits as a model (Arief et al., 2018). Adult Zebrafish display their complex brain structure and function, and importantly for epilepsy research, a endothelial blood-brain barrier and adult neurogenesis (Cho, Park, Baker & Reid, 2020).

Since most studies are still limited to zebrafish larvae research with adult Zebrafish is less established, and there are fewer humanization models available (Takai et al., 2020). Furthermore, in researching antiepileptic drugs researchers should be aware that Zebrafish might react differently to drugs than mammals as they lack some mammalian organs (lungs, mammary glands) (Cho, et al. 2017).

Mus musculus (Mouse)

While non-mammalian models have gained popularity in epilepsy research, the traditional mouse model is still used due to its complex behaviour and anatomy which gives this model high translational value (Turrini et al., 2017). With similar brain architecture and neurochemistry to humans, mice are well-suited for studying brain disorders such as epilepsy. One advantage of this model is that they display spontaneous recurrent seizures (Takai et al., 2020). Generally, mice models for epilepsy fall into one of two categories: genetic models or non-genetic models (which includes chemically induced models and electrically induced models) (Takai et al., 2020). 

For decades, researchers have been genetically modifying mice to create mutant strains, thus, there are numerous mouse models of specific types of epilepsy syndromes such as Dravet syndrome and Ohtahara syndrome (Fallah & Eubanks, 2020). Increasingly, researchers are opting to use ‘humanization’, replacing mouse DNA with the human orthologue, as these have greater relevance to human disease (Gawel et al., 2020). 

Since mice have been inbred for so long the differences in the genetic background among heterogeneous populations and inbred strains should be considered when interpreting data (Takai et al., 2020). Mice models also have the limitations of long maturation periods and an in utero development, making them unable to provide in vivo drug screening (Gawel et al., 2020).

Conclusion

Clinically and etiologically epilepsy is a very diverse condition, with various animal models being used to better understand the mechanisms behind the condition, and develop better drug therapeutics (Takai et al., 2020). Research into anti-epileptic drug (AEDs) development could affect many people’s lives, as currently, a third of people diagnosed with epilepsy find that the  AEDs on the market right now are ineffective at mitigating their seizures (Epilepsy Foundation of America, 2020). 

As we have discussed, each model organism being used in epilepsy research has different characteristics, which give them advantages and limitations in acting as a model for this condition. For example, the simplicity and availability of Drosophila and C. elegans mutants and RNAi lines make them ideal for genome-wide genetic screening whereas Mice and zebrafish are more suitable for studies of complex behavior (Takai et al., 2020). As a result, when used in conjunction, these models can provide new avenues for study that are not possible when constrained by the limitations of any one model. The simpler organisms (Drosophila and C. elegans) can also act as a preliminary model, offering an alternative to more complex models that is cheaper, lower maintenance, and has a shorter maturation time.

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