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.

项目总结/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，以及约三分之一的患者对抗癫痫发作无效的疾病 药物治疗在最严重的癫痫综合征的儿童，药物控制癫痫发作，甚至可以 更具挑战性。新的实验平台有可能在推进我们的研究方面发挥关键作用。 癫痫的治疗方法来源于人胚胎或诱导的脑类器官 多能干细胞就是这样一种具有巨大潜力的新技术。尤其如此 严重的儿童癫痫，因为类器官非常适合模拟早期神经发育。类器官 3D结构重现了人类大脑的复杂元素，如其层状组织和细胞 在人类大脑皮层的六层都有因为它们可以是人诱导多能干细胞（hiPSC）， 衍生的，类器官可以直接从患者组织产生。类器官技术的最新进展 已经导致产生不同的脑区域样类器官的能力，例如前脑皮层， 海马并形成抑制性和兴奋性细胞类型整合的“融合”结构。 在下面的提案中，我将利用这些进展，并建立在我拥有的一个类器官平台上。 最近开发的模型脑回路的形成和功能障碍的癫痫。以前，我可以 概括来自Rett综合征患者的类器官的超兴奋电图特征， 与癫痫发作和癫痫高度相关的神经系统疾病。我已经生成了大脑皮层 来自在SCN 8A基因中具有突变的hiPSC的海马类器官。该突变导致 严重的儿童癫痫。我发现SCN 8A突变体皮质类器官具有高度的超兴奋性， 与对照组相比，SCN 8A突变体海马缺乏生理活动模式，而SCN 8A突变体海马缺乏生理活动模式。 一种对记忆巩固很重要的特殊类型的神经振荡，称为尖波涟漪。这 这一发现表明，SCN 8A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 皮质中的兴奋性神经元与海马中的抑制性中间神经元。我现在将使用一个 技术，如钙指示剂成像、细胞外记录、免疫组织化学， 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。 假说.为了增加我的数据的严谨性和普遍性，我将使用来自三个不同患者的hiPSC 致病性SCN 8A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， 表型与观察到的表型相对应，并考虑作为患者的治疗剂。我希望这将 两者都为癫痫研究的新方法提供了蓝图，并加强了我们的治疗， 了解由SCN 8A突变引起的癫痫和神经回路功能障碍。