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

利用人脑类器官模拟 SCN8A 突变对皮层和海马的不同影响

• 批准号: 10624428
• 负责人: RANMAL A SAMARASINGHE
• 金额: $ 23.78万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10624428
• 关键词: 3-Dimensional; Anticonvulsants; Architecture; Biological Models; Biomedical Research; Brain; Brain region; Calcium; Cells; Child; Childhood; Complex; Data; Defect; Development; Disease; Drug usage; Electrodes; Elements; Embryo; Epilepsy; Etiology; Frequencies; Functional disorder; Generations; Goals; Hippocampus; Human; Image; Immunohistochemistry; Impaired cognition; Impairment; In Vitro; Individual; Interneurons; Medical; Memory impairment; Methodology; Modeling; Morphology; Mutation; Neural Network Simulation; Neurodevelopmental Disorder; Neurons; Organoids; Pathogenicity; Patients; Pattern; Phenotype; Physiological; Play; Pluripotent Stem Cells; Population; Prosencephalon; Public Health; Publishing; Refractory; Research; Rett Syndrome; Role; SCN8A gene; School-Age Population; Scientist; Seizures; Sodium Channel; Sorting; Structure; Subcellular Anatomy; Syndrome; System; Techniques; Technology; Testing; Therapeutic; Therapeutic Agents; Tissues; Training; Validation; Work; calcium indicator; career; cell type; childhood epilepsy; comparison control; drug testing; effective therapy; epileptic encephalopathies; epileptiform; excitatory neuron; extracellular; gain of function; gain of function mutation; genetic manipulation; genetic variant; human disease; human embryonic stem cell; in vivo; induced pluripotent stem cell; inhibitory neuron; insight; memory consolidation; migration; mutant; neonatal seizure; nervous system disorder; neural; neural circuit; neural model; neural network; neurodevelopment; neurophysiology; new technology; new therapeutic target; novel; novel therapeutics; programs; stem cell biology; synaptogenesis; therapeutic target; three dimensional structure; two-photon; voltage

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.

项目概要/摘要 癫痫是一种严重且使人衰弱的疾病，也是一个重大的公共卫生问题。 无法治愈的疾病，以及大约三分之一的患者对抗癫痫药物没有反应的疾病 对于最严重的儿童癫痫综合征，药物控制癫痫发作的效果可能会更好。 更具挑战性的新型实验平台有可能在推进我们的发展方面发挥关键作用。 源自人类胚胎或诱发的脑类器官的理解和治疗。 多能干细胞是一种具有巨大潜力的新技术。 严重的儿童癫痫，因为类器官非常适合模拟早期神经发育。 3D 结构概括了人脑的复杂元素，例如层状组织和细胞 在人类皮质的所有六层中都可以看到类型，因为它们可以是人类诱导多能干细胞（hiPSC）。 类器官可以直接从患者组织中产生，类器官技术的最新进展。 产生了产生独特的大脑区域类器官的能力，例如前脑皮层和 海马体并通过整合抑制性和兴奋性细胞类型来形成“融合”结构。 在下面的提案中，我将利用这些进步并建立在我拥有的类器官平台上 最近开发用于模拟癫痫的脑回路形成和功能障碍。 概括了来自雷特综合征患者的类器官的过度兴奋电图特征， 与癫痫发作和癫痫高度相关的神经系统疾病我现在已经产生了皮质和癫痫。 来自 hiPSC 的海马类器官含有 SCN8A 基因突变。 我发现 SCN8A 突变的皮质类器官具有过度兴奋性。 与对照组相比，SCN8A 突变体海马体的生理活动模式缺乏 对记忆巩固很重要的一种特殊类型的神经振荡，称为锐波波纹。 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 根据已发表的研究，我认为这种差异主要是由于功能障碍造成的。 我现在将使用一系列皮层中的兴奋性神经元与海马体中的抑制性中间神经元。 钙指示剂成像、细胞外记录、免疫组织化学等技术 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 为了提高数据的严谨性和普遍性，我将使用来自三个不同患者的 hiPSC。 最后，我将进行药物测试以进一步分离特定细胞的作用。 我预计这将是观察到的表型的类型并考虑作为患者的治疗剂。 两者都为癫痫研究的新方法提供了蓝图，并增强了我们的治疗和 了解 SCN8A 突变导致的癫痫和神经回路功能障碍。