Multi-scale disease modeling of SCN2A-related epilepsy due to gain-of-function variants

项目摘要癫痫影响了全球1%的人口，美国有300万人 独自一人。越来越多的儿童癫痫与离子通道基因的致病变异有关，包括 电压门控钠通道基因SCN2A。NINDS 2020年癫痫研究基准优先排序 确定基因变异如何导致癫痫和相关的神经发育障碍。SCN2A变种 表现为功能丧失与严重的神经发育障碍和迟发性癫痫有关。 另一方面，功能获得SCN2A变异体主要具有早发性癫痫的表型。 编码的钠通道(NaV1.2)在早期兴奋性谷氨酸能神经元中高表达 发展，为研究兴奋性神经元功能障碍如何导致早发性提供了一个独特的机会 癫痫。动物和人类组织衍生神经元模型为Dravet如何 综合征会导致中间神经元功能障碍和癫痫。在与SCN2A相关的疾病中，动物模型 说明功能丧失如何导致晚发性癫痫的自闭症谱系障碍。由于缺乏现成的 现有的疾病模型，对兴奋性神经元如何早期功能障碍的机制理解很少 在发育过程中会导致早发性癫痫。这项提案将利用两种早发性癫痫变种 临床表型趋同但生物物理机制不同的SCN2A型。患者来源的神经元 模型和鼠标模型提供了定义多个 尺度：从单个神经元到影响癫痫表型的神经回路。目标1将决定两个收益如何- 编码错义突变M1879T和E430A的功能缺失SCN2A变体通过以下方式增加兴奋性 不同的机制。分离和初级电路中IPSC来源神经元的功能分析将 明确不同的变异如何影响兴奋性，从而向癫痫表型趋同。目标2将 定义癫痫小鼠的海马区高级电路扰动，设计基因组编辑以 重述SCN2A-E430A人类癫痫性脑病。兴奋性变化的体外分析， 突触信号和海马区的网络输出将导致对如何获得功能的新的理解 SCN2A变异体会影响神经元网络。脑电和深度电极将提供时空关联 体内癫痫表型。这项提议将推动获奖者成为一名独立的内科科学家 结合了遗传性癫痫的多尺度建模方面的新专业知识，重点介绍了相关的教学方法，以及多样化的 专注于神经发育和遗传障碍的职业发展团队，所有这些都在高度合作的 培育低年级教师发展的环境。该奖项将提供1)定义特定变种的平台 自限性和顽固性癫痫对癫痫表型的贡献和2)研究如何进行靶向 未来R01资助的独立研究中，癫痫回路功能障碍影响回路输出和癫痫表型 研究。

Project Summary Epilepsy affects up to 1% of the population worldwide, and 3 million in the United States alone. A growing proportion of pediatric epilepsies are tied to causative variants in ion channel genes, including the voltage-gated sodium channel gene SCN2A. The 2020 Epilepsy Research Benchmarks of NINDS prioritize identifying how genetic variants cause epilepsy and related neurodevelopmental disorders. SCN2A variants that manifest with loss-of-function are associated with severe neurodevelopmental disorders and late-onset epilepsy. On the other hand, gain-of-function SCN2A variants predominantly have a phenotype of early-onset epilepsy. The encoded sodium channel (NaV1.2) is highly expressed in excitatory glutamatergic neurons early in development, presenting a unique opportunity to examine how excitatory neuron dysfunction leads to early-onset epilepsy. Animal and human tissue-derived neuron models have brought mechanistic insight to how Dravet syndrome results in interneuron dysfunction and epilepsy. Among SCN2A-related diseases, animal models illuminate how loss-of-function leads to autism spectrum disorder with late-onset epilepsy. Due to lack of readily available disease models, there is sparse mechanistic understanding of how excitatory neuron dysfunction early in development leads to early-onset epilepsy. This proposal will exploit two early-onset epilepsy variants of SCN2A that have a convergent clinical phenotype yet divergent biophysical mechanisms. Patient-derived neuron models and mouse models provide the opportunity to define the point of mechanistic convergence at multiple scales: from single neurons to neural circuits influencing epilepsy phenotype. Aim 1 will determine how two gain- of-function SCN2A variants, encoding missense mutations M1879T and E430A, confer increased excitability by distinct mechanisms. Functional analysis of iPSC-derived neurons in isolation and in elementary circuits will define how the different variants impact excitability and thus converge toward an epileptic phenotype. Aim 2 will define hippocampal higher-level circuit perturbations in epileptic mice designed with genome editing to recapitulate the SCN2A-E430A human epileptic encephalopathy. Ex vivo analysis of changes in excitability, synaptic signaling, and network output in the hippocampus will lead to new understanding of how gain-of-function SCN2A variants affect neuronal networks. EEG and depth electrodes will provide spatiotemporal correlate to the in vivo epilepsy phenotype. This proposal will propel the awardee to independence as a physician-scientist by incorporating new expertise in multi-scale modeling of genetic epilepsy, focused relevant didactics, and a diverse career development team specializing in neurodevelopmental and genetic disorders, all in a highly collaborative environment fostering junior faculty development. This award will provide a platform to 1) define variant-specific contributions to epilepsy phenotype in self-limited and intractable epilepsies and 2) investigate how targeted epileptic circuit dysfunction influences circuit output and epilepsy phenotype in future R01-funded independent research.