Department of Genetics, Center for Molecular Medicine, UMC Utrecht Brain Center, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
Department of Cardiology, Laboratory of Experimental Cardiology, University Medical Centre Leiden, Leiden, the Netherlands.
PLoS One. 2020 Mar 5;15(3):e0219106. doi: 10.1371/journal.pone.0219106. eCollection 2020.
Dravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human NaV1.1 or NaV1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous zebrafish models generated by random mutagenesis or morpholino oligomers. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the GABA antagonist pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the NaV1.1 activator AA43279 and two novel NaV1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of spontaneous burst movements and seizure activity. Our results show that selective inhibition of NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems for efficacy in mammals and to screen for potential side effects.
德拉维特综合征是由 SCN1A 的显性失活突变引起的，导致 Nav1.1 活性降低，从而导致神经元抑制不足。另一方面，SCN8A 的功能获得性突变可通过过度激活 NaV1.6 通道导致严重的癫痫性脑病亚型。这些观察结果表明，Nav1.1 和 Nav1.6 代表神经元抑制和激活之间平衡的两个对立方面。在这里，我们假设可以通过增强 Nav1.1 或降低 Nav1.6 活性来治疗德拉维特综合征。为了验证这一假设，我们生成并表征了一种新型 DS 斑马鱼模型，并测试了新的化合物，这些化合物分别选择性地激活或抑制人类 NaV1.1 或 NaV1.6 通道。我们使用 CRISPR/Cas9 生成了两个单独的 Scn1Lab 敲除系，作为以前通过随机诱变或 morpholino 寡聚物生成的斑马鱼模型的替代方法。使用优化的运动测定法，检测到仅在 Scn1Lab 敲除系中出现的自发爆发运动，并且当引入人类 SCN1A mRNA 时，该运动消失。除了行为表型外，Scn1Lab 敲除系还显示出大脑中的突发性电放电，表明斑马鱼中的癫痫发作。Scn1Lab 敲除系对 GABA 拮抗剂戊四氮的敏感性增加，并且整个生物体 GABA 水平降低。药物筛选进一步验证了德拉维特综合征表型。我们在 Scn1Lab 敲除系中测试了 NaV1.1 激活剂 AA43279 和两种新型 NaV1.6 抑制剂 MV1369 和 MV1312。这两种类型的化合物都显著减少了自发爆发运动和癫痫发作的次数。我们的结果表明，选择性抑制 NaV1.6 可能与选择性激活 NaV1.1 一样有效，这些方法可能成为治疗德拉维特综合征和其他（遗传性）癫痫的新潜在治疗策略。然而，在其他模型系统中，应进一步验证在斑马鱼中测试的化合物在哺乳动物中的疗效，并筛选潜在的副作用。
