Utilizing Human Brain Organoids to Model the Differential Effects of SCN8A Mutation on Cortex and Hippocampus

项目摘要/摘要 癫痫是一种严重的、使人衰弱的疾病，是一个重大的公共卫生问题。癫痫也是一种 没有药物治愈的疾病，以及大约三分之一的患者对抗癫痫药物没有反应的疾病 药物。在儿童时期最严重的癫痫综合征中，对癫痫发作的医学控制甚至可以 更具挑战性。新颖的实验平台有可能在推动我们的 对癫痫的认识和治疗。来源于人类胚胎或诱导的脑有机化合物 多能干细胞就是这样一项具有巨大潜力的新技术。这一点对于 严重的儿童期癫痫，因为有机化合物非常适合模拟早期神经发育。有机化合物是 3D结构概括了人脑的复杂元素，如大脑的层状组织和细胞 在人类大脑皮层的所有六层中都可以看到这种类型。因为它们可以是人类诱导多能干细胞(HiPSC) 衍生出来的有机化合物可以直接从病人的组织中产生。有机化合物技术的最新进展 导致了产生不同的大脑区域样器官的能力，如前大脑皮层和 并制造出融合了抑制性和兴奋性细胞类型的“融合”结构。 在下面的提案中，我将利用这些进步，并在我拥有的有机平台上构建 最近发展起来用来模拟癫痫的脑回路形成和功能障碍。此前，L能够 概述了来自Rett综合征患者的器官类物质的超兴奋性电信号特征。 与癫痫和癫痫高度相关的神经性疾病。我现在产生了大脑皮层和 来自具有SCN8A基因突变的HiPSCs的海马体。这种突变会导致 严重的儿童期癫痫。我发现SCN8A突变的皮质有机体具有高度的超兴奋性 与对照组相比，生理活动模式，而SCN8A突变的海马体缺乏 一种对巩固记忆很重要的特殊类型的神经振荡，称为尖波波动。这 研究结果表明，SCN8A突变导致不同大脑中不同的生理活动模式 地区。根据已发表的研究，我假设这种差异主要是由于大脑 皮层的兴奋性神经元与海马区的抑制性中间神经元。我现在将使用一个数组 钙指示剂成像、细胞外记录、免疫组织化学和 操纵类器官内兴奋性和抑制性神经元的遗传背景来测试这一点 假设。为了增加我的数据的严密性和概括性，我将使用来自三个不同患者的HiPSC 带有致病的SCN8A突变。最后，我将进行药物测试，以进一步分离特定细胞的作用 与观察到的表型相一致，并考虑作为患者的治疗剂。我期望这将会是 两者都为癫痫研究提供了一种新的方法蓝图，并加强了我们的治疗和 了解由SCN8A突变引起的癫痫和神经回路功能障碍。

Project Summary/Abstract Epilepsy is a severe and debilitating disease and a significant public health concern. Epilepsy is also a disease without a medical cure, and a disease where about 1 in 3 patients fails to respond to anti-seizure medications. In the most severe epilepsy syndromes of childhood, medical control of seizures can be even more challenging. Novel experimental platforms have the potential to play a critical role in advancing our understanding and treatment of epilepsy. Brain organoids derived from human embryonic or induced pluripotent stem cells are one such novel technology that has enormous potential. This is particularly true for severe childhood epilepsies, as organoids are ideally suited to model early neural development. Organoids are 3D structures that recapitulate complex elements of human brain such as its laminar organization and cell types seen in all six layers of human cortex. Since they can be human induced-pluripotent stem cell (hiPSC) derived, an organoid can be produced directly from patient tissue. Recent advances in organoid technology have resulted in the ability to generate distinct brain region-like organoids such as forebrain cortex and hippocampus and to make “fusion” structures with integration of inhibitory and excitatory cell types. In the following proposal I will leverage these advances and build on an organoid platform that I have recently developed to model brain circuit formation and dysfunction in epilepsy. Previously, l was able to recapitulate hyperexcitable electrographic features in organoids derived from a patient with Rett syndrome, a neurological disorder highly associated with seizures and epilepsy. I have now generated cortical and hippocampal organoids from hiPSCs harboring mutations in the SCN8A gene. This mutation results in a severe childhood epilepsy. I have found that the SCN8A mutant cortex organoids have a highly hyperexcitable pattern of physiological activity compared to controls, whereas the SCN8A mutant hippocampus lacks a particular type of neural oscillation that is important for memory consolidation called a sharp wave ripple. This finding suggests that the SCN8A mutation results in different physiological activity patterns in distinct brain regions. Based on published studies, I hypothesize that this difference is primarily due to dysfunction of excitatory neurons in the cortex versus inhibitory interneurons in the hippocampus. I will now use an array of techniques such as calcium indicator imaging, extracellular recordings, immunohistochemistry, and manipulation of the genetic background of excitatory and inhibitory neurons within the organoid to test this hypothesis. To increase the rigor and generalizability of my data, I will use hiPSC from three different patients with pathogenic SCN8A mutations. Finally, I will perform drug testing to further isolate the role of specific cell types to the observed phenotypes and for consideration as therapeutic agents in patients. I expect that this will both provide a blueprint for a novel methodology for epilepsy research and enhance our treatment and understanding of epilepsy and neural circuit dysfunction resulting from SCN8A mutations.