Inhibition of glutamate decarboxylase (GAD) by ethyl ketopentenoate (EKP) induces treatment-resistant epileptic seizures in zebrafish

Abstract

Epilepsy is a chronic brain disorder characterized by recurrent seizures due to abnormal, excessive and synchronous neuronal activities in the brain. It affects approximately 65 million people worldwide, one third of which are still estimated to suffer from refractory seizures. Glutamic acid decarboxylase (GAD) that converts glutamate into GABA is a key enzyme in the dynamic regulation of neural network excitability. Importantly, clinical evidence shows that lowered GAD activity is associated with several forms of epilepsy which are often treatment resistant. In the present study, we synthetized and explored the possibility of using ethyl ketopentenoate (EKP), a lipid-permeable GAD-inhibitor, to induce refractory seizures in zebrafish larvae. Our results demonstrate that EKP evoked robust convulsive locomotor activities, excessive epileptiform discharges and upregulated c-fos expression in zebrafish. Moreover, transgenic animals in which neuronal cells express apoaequorin, a Ca2+-sensitive bioluminescent photoprotein, displayed large luminescence signals indicating strong EKP-induced neuronal activation. Molecular docking data indicated that this proconvulsant activity resulted from the direct inhibition of both gad67 and gad65. Limited protective efficacy of tested anti-seizure drugs (ASDs) demonstrated a high level of treatment resistance of EKP-induced seizures. We conclude that the EKP zebrafish model can serve as a high-throughput platform for novel ASDs discovery.

Drug repurposing for Dravet syndrome in scn1Lab−/− mutant zebrafish

“…Clinical evidence shows that lowered GAD activity is associated with several forms of treatment-resistant epilepsy, and the model is considered to be relevant as a platform for novel antiepileptic drug discovery. 13 Conversely, the PTZ model, which conceptually builds on GABA A inhibition, is most sensitive to GABAergic AEDs (eg, barbiturates or benzodiazepines). 12 In general, the data showed good concordance between the three measurements.…”

“…Wild‐type zebrafish (AB strain) were used for experiments with PTZ and EKP. The Tg(elavl3:eGFP‐apoAequorin ) zebrafish line was used for the bioluminescence experiments …”

“…Similarly, Tg(elavl3:eGFP‐apoAequorin ) zebrafish larvae were preexposed to coelenterazine‐h (NanoLight Technology) and pretreated at 6 dpf with VHC or compound. Emitted photons were counted in a light‐tight thermostatted perfusion chamber at 7 dpf as described previously . For chemical induction of seizures, larvae were acutely exposed after compound pretreatment to 20 mmol·L –1 PTZ or 400 μmol·L –1 EKP …”

“…Emitted photons were counted in a light-tight thermostatted perfusion chamber at 7 dpf as described previously. 13 For chemical induction of seizures, larvae were acutely exposed after compound pretreatment to 20 mmol·L -1 PTZ 12 or 400 μmol·L -1 EKP. 13…”

“…Using the zebrafish DS model, our aim was to examine the possibility to repurpose these marketed medicines as AEDs, in particular in difficult to treat epilepsies such as DS. To examine further their potential broader antiseizure profile, we also tested the compounds in a pentylenetetrazol (PTZ) and a treatment‐resistant ethyl ketopentenoate (EKP) model in zebrafish. Our results show that LIS selectively decreased seizure activity in the DS zebrafish model, whereas EFA exhibited a broader antiseizure activity.…”