Zebrafish Models in Pre-Clinical Research
Zebrafish (Danio rerio) are one of the best leading models to study developmental biology, cancer, toxicology, drug discovery, and molecular genetics (Khan et al., 2018). Additionally, zebrafish are emerging as an important and useful model for studying neurodegenerative diseases generally, and ALS specifically (Babin et al., 2014).
Zebrafish offer unique advantages as a model system compared to higher vertebrates in general, and specifically in neurodegenerative diseases. The zebrafish genome is highly homologous to the human genome (Phillips & Westerfield, 2014), and genes implicated in neurodegenerative diseases are highly conserved between humans and zebrafish. Furthermore, they share all the same brain and CNS structures and cell types with humans. In addition, the large numbers of offspring and fast growing and optically transparent embryos enable rapid and statistically significant in vivo analysis. Moreover, zebrafish are efficient for toxin screening studies, and are relatively easy to manipulate using gene editing techniques (Ramesh et al., 2010).
Up until recently, mice, particularly the Sod1G93A model, have been the most common species used to model ALS (Hawrot et al., 2020; Cappella et al., 2019; Alrafih et al., 2018; Picher-Martel et al.,2016). However, to date, therapeutics that have been developed in genetic mouse models have failed to translate to effective interventions in humans, thus challenging the use of these models for therapeutic screening (Philips and Rothstein, 2015; Tosolini and Sleigh, 2017). One example is the repurposing of celecoxib for ALS in which treatment of Sod1G93A mice with celecoxib showed significant improvement in symptoms and survival (Drachman et al., 2002). However, a large Phase III study investigating the efficacy of celecoxib to treat ALS in humans failed (Cudkowicz et al., 2006). Additional examples of drugs that showed promise in treating ALS in mouse models, but failed to show efficacy in humans, are minocycline (Zhu et al., 2002; Gordon et al., 2007), creatine (Klivenyi et al., 1999; Groeneveld et al., 2003), and thalidomide (Kiaei, 2006; Stommel et al., 2009).
Taken together, the above data suggests that these models exist with significant caveats, such as modeling only particular aspects of the disease, developing non-ALS related phenotypes and a lack of translation to the clinic. Improved animal models need to be generated in order to better model the human disease (Philips and Rothstein, 2015).
Zebrafish have emerged as an important and useful vertebrate model for studying neurodegenerative diseases, including Parkinson’s disease (Unal et al., 2019; Vaz et al., 2018; Cronin et al., 2017), child epilepsy (Griffin et al., 2017) and ALS (Bose et al., 2019; Babin et al., 2014; Patten et al., 2014). Specifically, studies have shown that zebrafish models of Parkinson’s disease display conserved biochemical and neurobehavioral features of the phenomenology in humans. Furthermore, these models’ pharmacological response to drugs targeting the dopaminergic system used in patients in the clinic is conserved (e.g., rasagiline, quinpirole and SFK-38393 as dopamine agonists, and haloperidol and chlorpromazine as dopamine antagonists), suggesting that the underlying mechanisms that regulate movement are shared (Vaz et al., 2018).
In the case of epilepsy, when testing known anti-convalescent drugs on zebrafish with induced seizures, they caused an elevation in seizure latency, suggesting that the model can be used predictively for screening of potential anticonvulsive drugs (Gupta et al., 2018).
In addition, recent drug efficacy data from zebrafish was found to be convincing enough to continue directly to trials in humans, for instance in Dravet syndrome (a form of childhood epilepsy), where antiepileptic activity was shown in zebrafish carrying the disease-causing mutation (SCN1A) treated with lorcaserin, leading to treatment of five children with the disease, causing reduced seizure frequency and severity (Griffin et al., 2017). Efficacy data from zebrafish in the case of lymphatic anomalies also led to the treatment and improvement of a patient with a gain-of-function ARAF mutation (Li et al., 2019).
In regards to ALS, the zebrafish models carrying the common G93R SOD1 (Ramesh et al., 2010) and G348C TDP-43 (Schmid et al., 2013) mutations present hallmarks of adult onset neurodegenerative ALS (e.g., defective motor performance, loss of motor neurons, loss of neuromuscular connectivity and muscle atrophy). Within a few days from birth, the fish swim freely and when poked, dart away, making it easy to see and record motor defects (Ramesh et al., 2010). As such, zebrafish are a highly relevant model for studying ALS. Furthermore, when celecoxib, which showed promise in treating ALS in mouse models but failed to treat ALS in human clinical trials, was tested alone in zebrafish during the NeuroSense pre-clinical studies, it showed no effect (data not published), suggesting that the zebrafish model may prove to be more predictive in treating ALS than the mouse model. Also, when riluzole, one of only two commercial ALS drugs, was tested in the fish, they showed a slight improvement in their swimming capabilities, analogous to the minor effect in humans.
In view of the above experimental findings, we conclude that zebrafish is an ideal ALS model with which to test PrimeC.