Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.
Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.
J Neurosci Methods. 2018 Nov 1;309:132-142. doi: 10.1016/j.jneumeth.2018.09.004. Epub 2018 Sep 3.
Epilepsy affects around 1% of people, but existing antiepileptic drugs (AEDs) only offer symptomatic relief and are ineffective in approximately 30% of patients. Hence, new AEDs are sorely needed. However, a major bottleneck is the low-throughput nature of early-stage AED screens in conventional rodent models. This process could potentially be expedited by using simpler invertebrate systems, such as the nematode Caenorhabditis elegans.
Head-bobbing convulsions were previously reported to be inducible by pentylenetetrazol (PTZ) in C. elegans with loss-of-function mutations in unc-49, which encodes a GABA receptor. Given that epilepsy-linked mutations in human GABA receptors are well documented, this could represent a clinically-relevant system for early-stage AED screens. However, the original agar plate-based assay is unsuited to large-scale screening and has not been validated for identifying AEDs. Therefore, we established an alternative streamlined, higher-throughput approach whereby mutants were treated with PTZ and AEDs via liquid-based incubation.
Convulsions induced within minutes of PTZ exposure in unc-49 mutants were strongly inhibited by the established AED ethosuximide. This protective activity was independent of ethosuximide's suggested target, the T-type calcium channel, as a null mutation in the worm cca-1 ortholog did not affect ethosuximide's anticonvulsant action.
Our streamlined assay is AED-validated, feasible for higher throughput compound screens, and can facilitate insights into AED mechanisms of action.
Based on an epilepsy-associated genetic background, this C. elegans unc-49 model of seizure-like activity presents an ethical, higher throughput alternative to conventional rodent seizure models for initial AED screens.
癫痫影响约 1%的人群，但现有的抗癫痫药物（AEDs）仅提供症状缓解，并且在约 30%的患者中无效。因此，非常需要新的 AEDs。然而，一个主要的瓶颈是传统啮齿动物模型中早期 AED 筛选的低通量性质。通过使用更简单的无脊椎动物系统，如秀丽隐杆线虫，可以潜在地加速这一过程。
以前有报道称，unc-49 基因缺失功能突变的秀丽隐杆线虫在戊四氮（PTZ）作用下会诱导头摆动抽搐，unc-49 基因编码 GABA 受体。鉴于人类 GABA 受体的癫痫相关突变已有很好的记录，这可能代表了一个用于早期 AED 筛选的临床相关系统。然而，最初的琼脂平板测定法不适合大规模筛选，也没有经过验证用于识别 AEDs。因此，我们建立了一种替代的简化、高通量方法，通过液体孵育使突变体接受 PTZ 和 AED 处理。
unc-49 突变体在接触 PTZ 几分钟内诱导的抽搐被已建立的 AED 乙琥胺强烈抑制。这种保护作用与乙琥胺的建议靶点无关，即 T 型钙通道，因为线虫 cca-1 同源物的缺失突变不影响乙琥胺的抗惊厥作用。
我们的简化测定法经过 AED 验证，适用于高通量化合物筛选，并且可以促进对 AED 作用机制的深入了解。
基于与癫痫相关的遗传背景，这种秀丽隐杆线虫 unc-49 模型的类似癫痫发作的活动提供了一种伦理上的、更高通量的替代传统啮齿动物癫痫模型，用于初步 AED 筛选。
