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The etiology of epilepsy is a very complicated, multifactorial process that is not completely understood. Therefore, the availability of epilepsy animal models induced by different mechanisms is crucial in advancing our knowledge and developing new therapeutic regimens for this disorder. Considering the advantages of zebrafish, we have developed a seizure model in zebrafish larvae using ginkgotoxin, a neurotoxin naturally occurring in Ginkgo biloba and hypothesized to inhibit the formation of the neurotransmitter γ-aminobutyric acid (GABA). We found that a 2-hour exposure to ginkgotoxin induced a seizure-like behavior in zebrafish larvae. This seizure-like swimming pattern was alleviated by the addition of either pyridoxal-5′-phosphate (PLP) or GABA and responded quickly to the anti-convulsing activity of gabapentin and phenytoin, two commonly prescribed anti-epileptic drugs (AEDs). Unexpectedly, the ginkgotoxin-induced PLP depletion in our experimental setting did not affect the homeostasis of folate-mediated one-carbon metabolism, another metabolic pathway playing a crucial role in neural function that also relies on the availability of PLP. This ginkgotoxin-induced seizure behavior was also relieved by primidone, which had been tested on a pentylenetetrazole-induced zebrafish seizure model but failed to rescue the seizure phenotype, highlighting the potential use and complementarity of this ginkgotoxin-induced seizure model for AED development. Structural and morphological characterization showed that a 2-hour ginkgotoxin exposure did not cause appreciable changes in larval morphology and tissues development. In conclusion, our data suggests that this ginkgotoxin-induced seizure in zebrafish larvae could serve as an in vivo model for epileptic seizure research and potential AED screening.

A cDNA encoding for zebrafish γ-glutamyl hydrolase (γGH) was cloned and inserted into a pET43.1a vector via SmaI and EcoRI sites and expressed in Rosetta (DE3) cells as a Nus-His-tag fusion enzyme (NH-zγGH). After induction with isopropyl thiogalactoside, the enzyme was purified with a Ni-Sepharose column, and approximately 8 mg of pure enzyme was obtained per liter of culture. The primary sequence of the recombinant zγGH was similar to mammalian γGH. Thrombin digestion of this NH-zγGH fusion protein resulted in zγGH with approximately 2-fold higher catalytic activity compared with the NH-zγGH fusion enzyme. This recombinant zγGH is active and exhibits comparable endopeptidase activity with folate substrate and antifolate drug methotrexate. Use of this recombinant zγGH significantly increased efficiency in folylpolyglutamate hydrolysis for folate analysis compared with current protocols.

Dihydrofolate reductase (DHFR) catalyzes folic acid reduction and recycles dihydrofolate generated during dTMP biosynthesis to tetrahydrofolate. DHFR is the main target of methotrexate, the most widely used agent for antifolate therapy. Nevertheless, the emergence of methotrexate-resistance has greatly impeded the curative potential of this drug. Therefore, drugs with improved efficacy are still in demand, as well as an efficient in vitro assay system and animal model for antifolate drug discovery. The aim of this study is to evaluate the suitability of using zebrafish DHFR as an alternative assay system for antifolate drug discovery. The cDNAs encoding zebrafish and human DHFR were cloned, overexpressed, and purified. Similar structural and kinetic properties were revealed between zebrafish and human recombinant DHFRs. The susceptibilities of both enzymes to known DHFR inhibitors, including methotrexate and trimethoprim, and compounds with antifolate potential, such as polyphenols, are also comparable. In addition, the DHFR-mediated dihydrofolate reduction was significantly inhibited by its own substrate folic acid. An unexpected tissue-specific distribution of DHFR was observed with the highest level present in ova and brains of zebrafish. DHFR is also abundant in zebrafish embryos of early stages and decreased abruptly after 3 days postfertilization. The substantial resemblance between zebrafish and human DHFRs, as demonstrated in this study, provides compelling evidence supporting the use of zebrafish DHFR as an in vitro assay system for folate-related studies and drug discovery.