Zebrafish as a robust preclinical platform for screening plant-derived drugs with anticonvulsant properties—a review

Traditionally, selected plant sources have been explored for medicines to treat convulsions. This continues today, especially in countries with low-income rates and poor medical systems. However, in the low-income countries, plant extracts and isolated drugs are in high demand due to their good safety profiles. Preclinical studies on animal models of seizures/epilepsy have revealed the anticonvulsant and/or antiepileptogenic properties of, at least some, herb preparations or plant metabolites. Still, there is a significant number of plants known in traditional medicine that exert anticonvulsant activity but have not been evaluated on animal models. Zebrafish is recognized as a suitable in vivo model of epilepsy research and is increasingly used as a screening platform. In this review, the results of selected preclinical studies are summarized to provide credible information for the future development of effective screening methods for plant-derived antiseizure/antiepileptic therapeutics using zebrafish models. We compared zebrafish vs. rodent data to show the translational value of the former in epilepsy research. We also surveyed caveats in methodology. Finally, we proposed a pipeline for screening new anticonvulsant plant-derived drugs in zebrafish (“from tank to bedside and back again”)

Discovery of (R)-N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide [(R)-AS-1], a novel orally bioavailable EAAT2 modulator with drug-like properties and potent antiseizure activity in vivo

[Image: see text] (R)-7 [(R)-AS-1] showed broad-spectrum antiseizure activity across in vivo mouse seizure models: maximal electroshock (MES), 6 Hz (32/44 mA), acute pentylenetetrazol (PTZ), and PTZ-kindling. A remarkable separation between antiseizure activity and CNS-related adverse effects was also observed. In vitro studies with primary glia cultures and COS-7 cells expressing the glutamate transporter EAAT2 showed enhancement of glutamate uptake, revealing a stereoselective positive allosteric modulator (PAM) effect, further supported by molecular docking simulations. (R)-7 [(R)-AS-1] was not active in EAAT1 and EAAT3 assays and did not show significant off-target activity, including interactions with targets reported for marketed antiseizure drugs, indicative of a novel and unprecedented mechanism of action. Both in vivo pharmacokinetic and in vitro absorption, distribution, metabolism, excretion, toxicity (ADME-Tox) profiles confirmed the favorable drug-like potential of the compound. Thus, (R)-7 [(R)-AS-1] may be considered as the first-in-class small-molecule PAM of EAAT2 with potential for further preclinical and clinical development in epilepsy and possibly other CNS disorders

SCN1A overexpression, associated with a genomic region marked by a risk variant for a common epilepsy, raises seizure susceptibility

Mesial temporal lobe epilepsy with hippocampal sclerosis and a history of febrile seizures is associated with common variation at rs7587026, located in the promoter region of SCN1A. We sought to explore possible underlying mechanisms. SCN1A expression was analysed in hippocampal biopsy specimens of individuals with mesial temporal lobe epilepsy with hippocampal sclerosis who underwent surgical treatment, and hippocampal neuronal cell loss was quantitatively assessed using immunohistochemistry. In healthy individuals, hippocampal volume was measured using MRI. Analyses were performed stratified by rs7587026 type. To study the functional consequences of increased SCN1A expression, we generated, using transposon-mediated bacterial artificial chromosome transgenesis, a zebrafish line expressing exogenous scn1a, and performed EEG analysis on larval optic tecta at 4 day post-fertilization. Finally, we used an in vitro promoter analysis to study whether the genetic motif containing rs7587026 influences promoter activity. Hippocampal SCN1A expression differed by rs7587026 genotype (Kruskal-Wallis test P = 0.004). Individuals homozygous for the minor allele showed significantly increased expression compared to those homozygous for the major allele (Dunn's test P = 0.003), and to heterozygotes (Dunn's test P = 0.035). No statistically significant differences in hippocampal neuronal cell loss were observed between the three genotypes. Among 597 healthy participants, individuals homozygous for the minor allele at rs7587026 displayed significantly reduced mean hippocampal volume compared to major allele homozygotes (Cohen's D = - 0.28, P = 0.02), and to heterozygotes (Cohen's D = - 0.36, P = 0.009). Compared to wild type, scn1lab-overexpressing zebrafish larvae exhibited more frequent spontaneous seizures [one-way ANOVA F(4,54) = 6.95 (P < 0.001)]. The number of EEG discharges correlated with the level of scn1lab overexpression [one-way ANOVA F(4,15) = 10.75 (P < 0.001]. Finally, we showed that a 50 bp promoter motif containing rs7587026 exerts a strong regulatory role on SCN1A expression, though we could not directly link this to rs7587026 itself. Our results develop the mechanistic link between rs7587026 and mesial temporal lobe epilepsy with hippocampal sclerosis and a history of febrile seizures. Furthermore, we propose that quantitative precision may be important when increasing SCN1A expression in current strategies aiming to treat seizures in conditions involving SCN1A haploinsufficiency, such as Dravet syndrome

A Review on the Role and Function of Cinnabarinic Acid, a “Forgotten” Metabolite of the Kynurenine Pathway

In the human body, the majority of tryptophan is metabolized through the kynurenine pathway. This consists of several metabolites collectively called the kynurenines and includes, among others, kynurenic acid, L-kynurenine, or quinolinic acid. The wealth of metabolites, as well as the associated molecular targets and biological pathways, bring about a situation wherein even a slight imbalance in the kynurenine levels, both in the periphery and central nervous system, have broad consequences regarding general health. Cinnabarinic acid (CA) is the least known trace kynurenine, and its physiological and pathological roles are not widely understood. Some studies, however, indicate that it might be neuroprotective. Information on its hepatoprotective properties have also emerged, although these are pioneering studies and need to be replicated. Therefore, in this review, I aim to present and critically discuss the current knowledge on CA and its role in physiological and pathological settings to guide future studies

N-Benzyl-(2,5-dioxopyrrolidin-1-yl)propanamide (AS-1) with Hybrid Structure as a Candidate for a Broad-Spectrum Antiepileptic Drug

In our recent studies, we identified compound N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide (AS-1) as a broad-spectrum hybrid anticonvulsant which showed potent protection across the most important animal acute seizure models such as the maximal electroshock (MES) test, the subcutaneous pentylenetetrazole (s.c. PTZ) test, and the 6-Hz (32 mA) test in mice. Therefore, AS-1 may be recognized as a candidate for new anticonvulsant effective in different types of human epilepsy with a favorable safety margin profile determined in the rotarod test in mice. In the aim of further pharmacological evaluation of AS-1, in the current study, we examined its activity in the 6-Hz (44 mA) test, which is known as the model of drug-resistant epilepsy. Furthermore, we determined also the antiseizure activity in the kindling model of epilepsy induced by repeated injection of pentylenetetrazole (PTZ) in mice. As a result, AS-1 revealed relatively potent protection in the 6-Hz (44 mA) test, as well as delayed the progression of kindling induced by repeated injection of PTZ in mice at doses of 15 mg/kg, 30 mg/kg, and 60 mg/kg. Importantly, the isobolographic analysis showed that a combination of AS-1 and valproic acid (VPA) at the fixed ratio of 1:1 displayed a supra-additive (synergistic) interaction against PTZ-induced seizures in mice. Thus, AS-1 may be potentially used in an add-on therapy with VPA. Moreover, incubation of zebrafish larvae with AS-1 substantially decreased the number, cumulative but not the mean duration of epileptiform-like events in electroencephalographic assay. Finally, the in vitro ADME-Tox studies revealed that AS-1 is characterized by a very good permeability in the parallel artificial membrane permeability assay test, excellent metabolic stability on human liver microsomes (HLMs), no significant influence on CYP3A4/CYP2D6 activity, and moderate inhibition of CYP2C9 in a concentration of 10 μM, as well as no hepatotoxic properties in HepG2 cells (concentration of 10 μM)

Zebrafish Larvae Carrying a Splice Variant Mutation in cacna1d: A New Model for Schizophrenia-Like Behaviours?

Abstract Persons with certain single nucleotide polymorphisms (SNPs) in the CACNA1D gene (encoding voltage-gated calcium channel subunit alpha 1-D) have increased risk of developing neuropsychiatric disorders such as bipolar, schizophrenia and autism. The molecular consequences of SNPs on gene expression and protein function are not well understood. Thus, the use of animal models to determine genotype-phenotype correlations is critical to understanding disease pathogenesis. Here, we describe the behavioural changes in larval zebrafish carrying an essential splice site mutation ( sa17298 ) in cacna1da . Heterozygous mutation resulted in 50% reduction of splice variants 201 and 202 (haploinsufficiency), while homozygosity increased transcript levels of variant 201 above wild type (WT; gain-of-function, GOF). Due to low homozygote viability, we focused primarily on performing the phenotypic analysis on heterozygotes. Indeed, cacna1da sa17298/WT larvae displayed hyperlocomotion—a behaviour characterised in zebrafish as a surrogate phenotype for epilepsy, anxiety or psychosis-like behaviour. Follow-up tests ruled out anxiety or seizures, however, as neither thigmotaxis defects nor epileptiform-like discharges in larval brains were observed. We therefore focused on testing for potential “psychosis-like” behaviour by assaying cacna1da sa17298/WT larval locomotor activity under constant light, during light-dark transition and in startle response to dark flashes. Furthermore, exposure of larvae to the antipsychotics, risperidone and haloperidol reversed cacna1da -induced hyperactivity to WT levels while valproate decreased but did not reverse hyperactivity. Together, these findings demonstrate that cacna1da haploinsufficiency induces behaviours in larval zebrafish analogous to those observed in rodent models of psychosis. Future studies on homozygous mutants will determine how cacna1d GOF alters behaviour in this context

Neuroprotective Properties of Oleanolic Acid—Computational-Driven Molecular Research Combined with In Vitro and In Vivo Experiments

Oleanolic acid (OA), as a ubiquitous compound in the plant kingdom, is studied for both its neuroprotective and neurotoxic properties. The mechanism of acetylcholinesterase (AChE) inhibitory potential of OA is investigated using molecular dynamic simulations (MD) and docking as well as biomimetic tests. Moreover, the in vitro SH-SY5Y human neuroblastoma cells and the in vivo zebrafish model were used. The inhibitory potential towards the AChE enzyme is examined using the TLC-bioautography assay (the IC50 value is 9.22 μM). The CH-π interactions between the central fragment of the ligand molecule and the aromatic cluster created by the His440, Phe288, Phe290, Phe330, Phe331, Tyr121, Tyr334, Trp84, and Trp279 side chains are observed. The results of the in vitro tests using the SH-SY5Y cells indicate that the viability rate is reduced to 71.5%, 61%, and 43% at the concentrations of 100 µg/mL, 300 µg/mL, and 1000 µg/mL, respectively, after 48 h of incubation, whereas cytotoxicity against the tested cell line with the IC50 value is 714.32 ± 32.40 µg/mL. The in vivo tests on the zebrafish prove that there is no difference between the control and experimental groups regarding the mortality rate and morphology (p > 0.05)

Developmental Exposure to Kynurenine Affects Zebrafish and Rat Behavior

Proper nutrition and supplementation during pregnancy and breastfeeding are crucial for the development of offspring. Kynurenine (KYN) is the central metabolite of the kynurenine pathway and a direct precursor of other metabolites that possess immunoprotective or neuroactive properties, with the ultimate effect on fetal neurodevelopment. To date, no studies have evaluated the effects of KYN on early embryonic development. Thus, the aim of our study was to determine the effect of incubation of larvae with KYN in different developmental periods on the behavior of 5-day-old zebrafish. Additionally, the effects exerted by KYN administered on embryonic days 1–7 (ED 1–7) on the behavior of adult offspring of rats were elucidated. Our study revealed that the incubation with KYN induced changes in zebrafish behavior, especially when zebrafish embryos or larvae were incubated with KYN from 1 to 72 h post-fertilization (hpf) and from 49 to 72 hpf. KYN administered early during pregnancy induced subtle differences in the neurobehavioral development of adult offspring. Further research is required to understand the mechanism of these changes. The larval zebrafish model can be useful for studying disturbances in early brain development processes and their late behavioral consequences. The zebrafish-medium system may be applicable in monitoring drug metabolism in zebrafish

Anticonvulsant Activity of Pterostilbene in Zebrafish and Mouse Acute Seizure Tests

Pterostilbene (PTE), a natural dimethylated analog of resveratrol, possesses numerous health-beneficial properties. The ability of PTE to cross the blood–brain barrier raised the possibility that this compound may modulate central nervous system functions, including seizure activity. The aim of our study was to investigate the activity of PTE in the larval zebrafish pentylenetetrazole (PTZ) seizure assay and three acute seizure tests in mice, i.e., in the maximal electroshock seizure threshold (MEST), 6 Hz-induced psychomotor seizure threshold and intravenous (iv) PTZ tests. Additionally, potential antidepressant activity of PTE was estimated in the forced swim test in mice. The chimney test was used to determine the influence of PTE on motor coordination in mice, while its influence on neuromuscular strength was assessed in the grip strength test in mice. Locomotor activity was determined to verify the results from the forced swim test. PTE revealed an evident anticonvulsant effect both in zebrafish larvae (10 µM; 2 h-incubation) and mice (at doses of 100 and 200 mg/kg, intraperitoneally) but it did not exhibit antidepressant potential in the forced swim test. Furthermore, it did not cause any statistically significant changes in motor coordination, neuromuscular strength and locomotor activity in mice. In conclusion, our present findings demonstrate for the first time the anticonvulsant potential of PTE. The aforementioned results suggest that it might be employed in epilepsy treatment, however, further precise studies are required to verify its activity in other experimental seizure and epilepsy models and its precise mechanism of action should be determined